STEREO-CITE
PROTEO-TRANSCRIPTOMICS SETUSER MANUAL

Cat. No.: 211PT114(4 RXNs)
Kit Version: V1.0
Manual Version: B

STUM-TT005

STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

REVISION HISTORY
Manual Version:
Kit Version:
Date:
Description：

A
V1.0
Mar. 2024
Initial release

Manual Version:
Kit Version:
Date:

B
V1.0
May. 2024

Description：

•
•
•
•
•
•
•

Typo fixation
Addition of compatible sequencing platforms in
Section 1.2 and Chapter 6
Addition of recommended antibody dilution in Section
4.3
Addition of operational instruction for specific types oftissue in Section 4.5
Addition of operational instruction for using the same
PCR Thermal Cycler for permeabilization and reverse
transcription steps in Section 4.7 and 4.8
Removal of operational instruction for keeping the chipmoist when removing solution in Section 4.7
Modification of operational instruction for keeping thebeads and discard the supernatant in step b. of Section
5.5

STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

Note: Please download the latest version of the manual and use it with thecorresponding Stereo-seq Transcriptomics T kit.
©2024 STOmics Tech Co., Ltd. at Shenzhen.
All rights reserved.
1. The products shall be for research use only, not for use in diagnostic procedures.
2. The Content on this manual may be protected in whole or in part by applicable
intellectual property laws. STOmics Tech Co., Ltd. and/or corresponding right subjects
own their intellectual property rights according to law, including but not limited to
trademark rights, copyrights, etc.
3. STOmics Tech Co., Ltd. do not grant or imply the right or license to use any copyrighted
content or trademark (registered or unregistered) of us or any third party. Without ourwritten consent, no one shall use, modify, copy, publicly disseminate, change, distribute,or publish the program or Content of this manual without authorization, and shall not
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4. Nothing contained herein is intended to or shall be construed as any warranty,
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Any and all warranties applicable to any products listed herein are set forth in theapplicable terms and conditions of sale accompanying the purchase of such product.
STOmics Tech Co., Ltd., Shenzhen makes no warranty and hereby disclaims any and all
warranties as to the use of any third-party products or protocols described herein.

STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

WORKFLOW

STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

TABLE OF CONTENTS
CHAPTER 1: INTRODUCTION
1.1.
Intended Use							1.2. Sequencing Guideline				1.3. List of Kit Components 				1.4. Additional Equipment and Materials		
1.5. Practice Tips 					
1.6. Precautions and Warnings 			

1
1
1
5
9
13

CHAPTER 2: SAMPLE AND EXPERIMENTAL PREPARATIONCHAPTER 3: PILOT STEREO-CITE EXPERIMENT
3.1. Experimental Preparation					173.2. Cryosection Preparation					193.3. Tissue Mounting on Microscopic Slides			20
3.4. Tissue Fixation 							213.5. Tissue Blocking and Primary Antibody Incubation
22
3.6. Secondary Antibody Incubation				
23
3.7. Imaging				24
3.8. Stereo-CITE Pilot Experiment Results			
25
CHAPTER 4: Stereo-CITE PROTEO-TRANSCRIPTOMICS SETSTANDARD OPERATING PROCEDURE
4.1. Experimental Preparation					274.2. Tissue Fixation							314.3. Tissue Blocking and Primary Antibody Incubation
31
4.4. DAPI Staining							344.5. Imaging								364.6. Tissue Decrosslinking						384.7. Reverse Transcription 			
39
4.8. Tissue Removal						414.9. cDNA & ADT Release and Collection			41
4.10. cDNA Purification and Amplification			42
4.11. ADT Product Purification					
43STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

TABLE OF CONTENTS
CHAPTER 5: LIBRARY PREPARATION
5.1. Experimental Preparation					5.2. cDNA Fragmentation and Amplification			5.3. PCR Product Size Selection					5.4. ADT Product Amplification					5.5. ADT PCR Product Size Selection				

56
56
58
59
61

CHAPTER 6: LIBRARY CONSTRUCT & SEQUENCING		

62

APPENDIX
Appendix A: PCR Barcode Primer Mix Use Rules
Appendix B: 16 PCR Barcode Primer Mix Use Rules
Appendix C: Antibody Titration				

65
66
68

NOTE: Additional operation tips and guidance.
CRITICAL STEPS: Pay extra attention for these steps to avoid experimental setbacks or
problematic results.
QUALITY CHECK POINT
CAUTION: Proceed with extra care; improper handling or carelessness may cause
experimental failure or accidents.
STOP POINT: Here you may pause your experiment and store your sample.

CHAPTER 1
INTRODUCTION

STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

1.1. Intended Use
STOmics Stereo-CITE Proteo-Transcriptomics Set is intended to simultaneously detect
the whole transcriptome and high-plex protein on the same tissue section. Built upon
DNA Nanoball (DNB) technology, STOmics Stereo-CITE Proteo-Transcriptomics Set
enables a “tissue-to-data” solution through in situ capture of the whole transcriptome
and high-plex protein markers, at nanoscale resolution and centimeter-sized field ofview. The Stereo-seq Chip T (poly-T-based chip) is loaded with capture probes containing
spatial coordinate information. Through a series of biochemical processes, the probes
can capture mRNA molecules and antibody-derived tags (ADTs) in situ within the tissue,
then through cDNA synthesis, and obtain transcriptome plus multi-protein spatial
distribution information of the entire tissue through sequencing and a complementary
visualization platform.
All reagents provided in this kit have passed stringent quality control and functionalverification, ensuring performance stability and reproducibility.

1.2. Sequencing Guideline
Sequencing libraries produced via the Stereo-CITE Proteo-Transcriptomics Set requires
the DNBSEQ sequencing platform.
Compatible sequencing platforms:• DNBSEQ-G400·Pooling both Stereo-CITE transcriptome and ADT libraries on the same flowcell is notrecommended.
·It is recommended to sequence Stereo-CITE transcriptome and ADT libraries
separately on different flowcells.
• DNBSEQ-T7RS·Pooling both Stereo-CITE transcriptome and ADT libraries on the same flowcell isrecommended.
For details refer to Chapter 6: Library Construct and Sequencing of this manual.

1.3. List of Kit Components
Each Stereo-seq Transcriptomics Set for Chip-on-a-slide consists of:•

Stereo-seq Transcriptomics T Kit *1 (4 RXN)•

Stereo-seq Chip T Slide (1cm*1cm) *1 (4 EA)•

STOmics Stereo-seq Accessory Kit *2 (5 PCs)•

Stereo-seq Protein Assisted Kit *1 (4 RXN)

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1. INTRODUCTION

STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

The Stereo-seq Library Preparation Kit is not included in the Stereo-seq Transcriptomics
Set for Chip-on-a-slide kit and needs to be purchased separately. If you wish toconstruct a Stereo-seq library in-house refer to Chapter 5: Library Preparation for moredetail.

Compatible auxiliary not included:•

Stereo-seq PCR Adaptor *1 (2 EA)

Further information on catalog numbers, kit components, and specifications are listed
below (next page).

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1. INTRODUCTION

STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

Upon receiving the Stereo-seq Chip T Slide (1cm*1cm), follow the instructionsin Stereo-seq Chip P Slide_Stereo-seq Chip T Slide Operation Guide For Receiving,
Handling And Storing to properly store unused Stereo-seq Chip T Slides.
Performance of products may only be guaranteed before their expiration date.
Proper performance is also subject to the products being transported, stored, andused in appropriate conditions.

Table 1-1 Stereo-seq Transcriptomics T Kit

Stereo-seq Transciptomics T Kit

Cat. No.:111KT114

Component

Reagent Cat. No.

RI

1000028499

300 µL

×1

PR Enzyme

1000028500

10 mg

×1

PR Rinse Buffer

1000042897

880 µL

×1

Glycerol

1000031615

50 µL

×1

RT Reagent

1000042898(transparent)

720 µL×1

RT Oligo

1000028508(transparent)

1 OD

×1

RT Additive

1000028502(transparent)

44 µL

×1

ReverseT Enzyme

1000042899(transparent)

44 µL

×1

TR Buffer

1000028505

1725 µL

×2

cDNA Release Enzyme 1000028511

88 µL

×1

cDNA Release Buffer

1000028512

1725 µL

×2

cDNA Primer

1000028513

36 µL

×1

cDNA Amplification
Mix

1000028514

220 µL

×1

Storage Temperature:-25°C~-18°C

Cap Color

Transported
by cold chain

3

Quantity (tube)

Expiration Date:refer to label

1. INTRODUCTION

STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

Table 1-2 Stereo-seq Chip T Slide (1cm*1cm)

Stereo-seq Chip T Slide (1cm*1cm)

Cat. No.: 210CT114

Component

Reagent Cat. No.

Cap Color

Quantity (per kit)

Stereo-seq Chip T(1cm * 1cm)

-

-

4 EA

Transported
by cold chain

Storage Temperature:-25°C~8°C

Expiration Date:refer to label

Table 1-3 Stereo-seq Protein Assisted Kit

Stereo-seq Protein Assisted Kit

Cat. No.: 212KA114

Component

Reagent Cat. No.

Cap Color

Decrosslinking
Reagents

1000043549

1725 µL x 2

ADT Amplification Kit 1000043547

220 µL x 1

ADT Primer Mix

1000043548

36 µL x 1

Blocking Reagents

1000044666(transparent)
Transported
by cold chain

Storage Temperature:-25°C~ -18°C

Quantity (per kit)

60 µL x 1
Expiration Date:refer to label

Table 1-4 STOmics Accessory Kit

STOmics Accessory Kit

Cat. No.: 1000033700

Component

Reagent Cat. No.

Cap Color

Quantity (per kit)

Cassette

1000033699

-

1 EA

Gasket1000033698

-

4 EA

Sealing Tape

1000042970

-

6 EA

Storage Temperature:
Room Temperature

Transported atroom temperature

Expiration Date:refer to label

Table 1-5 Stereo-seq PCR Adaptor

Stereo-seq PCR Adaptor

Cat. No.: 301AUX001

Component

Reagent Cat. No.

Cap Color

Quantity (per kit)

Stereo-seq PCR
Adaptor

-

-

2 EA

Storage Temperature:
Room Temperature

Transported atroom temperature

4

Expiration Date:refer to label
1. INTRODUCTION

STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

1.4. Additional Equipment and Materials
The tables below list equipment and materials needed for this protocol. Some common
laboratory equipments not named in Table 1-6 are expected to be accessible by the user;
for instance, an ice maker, biological safety cabinet, freezers, and so on. For specificmicroscope requirements, refer to STOmics Microscope Assessment Guideline.
Table 1-6 Additional equipment required

Equipment
Brand

Description

Cat. No.

-

Cryostat--Benchtop centrifuge--

Pipettes--

pH meter-

Metal heating block dry bath (optional)--

Vortex mixer-

Bio-Rad*T100 Thermal Cycler

1861096

ABI*
LabnetProFlex 3 x 32-well PCR System
Slide Spinner (optional)

4483636
C1303-T

NEBNext®

Magnetic Separation Rack for <200 µL
tubes

S1515S

Magnetic rack DynaMagTM-2 for 1.5-2mL
tubes

12321D

Qubit™3 fluorometer

Q33216

Agilent 2100 bioanalyzer

G2939AA (or similar)

Thermo Fisher
Scientific
Agilent
Technologies™

Choose either one of the listed brands (with * mark). Suitable PCR adaptor will beneeded.

Reagents
Brand

Description

Cat. No.

-

100% Ethanol (Analytical grade)

-

Gibco

PBS (10X), pH 7.4 - for PFA fixed tissue

70011044

BOSTER (or otherbrands)4% Paraformaldehyde (with DEPC) - for
PFA fixed tissue

AR1069

Nuclease-free water

AM9937

20X SSCAM9770

1X TE buffer, pH 8.0

AM9858Ambion5

1. INTRODUCTION

STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual*Beckman Coulter*VAZYME
Sigma Aldrich
SAKURA
Invitrogen
Agilent
Technologies™

Thermo Fisher
Scientific™

AMPure® XP

A63882SPRIselect

B23317/B23318/
B23319VAHTSTM DNA Clean Beads

N411-02

Hydrochloric acid, HCl (0.1 N)

2104-50ML

Methanol

34860-1L-R

SAKURA Tissue-Tek® O.C.T. Compound

4583

Salmon Sperm DNA (1mg/mL)

AM9680Qubit dsDNA HS Assay Kit

Q32854High sensitivity DNA kit

5067-4626

High sensitivity RNA kit

5067-1513

DAPI Solution (1 mg/mL)

62248

Gibco™ Horse Serum*

26050070

Gibco™ Goat Serum*

16210064

Alexa Fluor™ Plus anti-Rat secondary
Antibody (for mouse samples)

A48270Alexa Fluor™ Plus anti-Mouse secondary
Antibody (for human samples)

A32773

Other fluorescent secondary antibodies should be tested before use.
TotalSeq-A™ antibody

BioLegend

TotalSeq-A™ Mouse Universal Cocktail,V1.0
TotalSeq-A™ Human Universal Cocktail,V1.0
Human TruStain FcX™ (Fc Receptor
Blocking Solution)

199901399907422301

TruStain FcX™ PLUS (anti-mouse CD16/32)
156604
Antibody
FcR Blocking Reagent is for blocking of Fc receptors on the cell membrane. Please
select either one according to the host species of your sample. Please choose
Human TruStain FcX™ (Fc Receptor Blocking Solution) for human tissues andchoose TruStain FcX™ PLUS (anti-mouse CD16/32) Antibody for mouse tissues.
Choose either one of the listed brands (with * mark). Suitable PCR adaptor will beneeded.
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1. INTRODUCTION

STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

Stereo-seq Transcriptomics T Kit has been validated to be compatible with thesingular TotalSeq-A™ antibody mixture, the TotalSeq-A™ Mouse, and Human
Universal Cocktails from Biolegend. Please feel free to combine the TotalSeq-A™
antibody depending on your experimental design. All the TotalSeq-A™ antibodies
to be used must have their optimal concentrations determined by titration before
combination. Please find more information in Appendix C. For detailed
information, please refer to the Biolegend website: https://www.biolegend.com/
en-us/search-results?PageSize=25&Format=TOTALS EQ_A

Special Notification:
Isotype control antibody
An isotype control antibody has similar characteristics to the primary antibody
but lacks specificity for the target of interest. Isotype control antibodies are often
used as negative controls to help distinguish non-specific background signals from
specific antibody signals. The selection of an isotype control antibody should matchthe species and class of the primary antibody, including the light chain.
For example, in Figure 1, the TotalSeq™-A0007 anti-human CD274 (B7-H1, PD-L1)
antibody has a mouse IgG2b κ isotype. Therefore, when detecting this antibody, it isnecessary to include the TotalSeq™-A0092 Mouse IgG2b, κ isotype control antibody.
The specific type of isotype control antibody to add should be determined based on
the type of TotalSeq™-A primary antibody used. Additionally, it is important to notethat if the kit is used with TotalSeq™-A Mouse Universal Cocktail, V1.0 (Biolegend,Cat. No. 199901) or TotalSeq™-A Human Universal Cocktail, V1.0 (Biolegend, Cat.
No. 399907), there is no need to add an isotype control antibody as these cocktails
already include isotype control antibodies.
The selection of isotype control antibodies can be found at this website:
https://www.biolegend.com/en-us/search-results?PageSize=25&Category=ISO_CTRL&Format=TOTALSEQ_A

7

1. INTRODUCTION

STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

Consumables
Brand

Description

Cat. No.

-

Stainless-steel base mold--

Aluminum foil--

Forceps--

Slide staining rack-

Sterilized Syringe-

Slide Container-

Microscope Slide Storage Box/IHC wet box Microscope Slides
Millipore-

Super PAP Pen (hydrophobic barrier pen) SLGV033N
Millex Syringe Filter, Durapore PVDF, 0.22
µm pore size-

Microscope glass coverslip (area: 18 mm x
18 mm, thickness: 0.13 - 0.16 mm)
Corning® 100 mm TC-treated Culture Dish 353003

Corning

50 mL centrifuge tubes

43082915 mL centrifuge tubes

430791Kimtech

KimWipesTM delicate task wipes

34155

MATIN

Power Dust Remover (can be substituted
with other similar power dust remover)M-6318

1.5 mL centrifuge tubes

MCT-150-A

0.2 mL PCR tubes*

PCR-02-C

96-well PCR plate*PCR-96M2-HS-C

1,000 µL filtered tips

TF-1000-L-R-S

200 µL filtered tips

TF-200-L-R-S

100 µL filtered tips

TF-100-R-S

Axygen8

1. INTRODUCTION

STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

10 µL filtered tips

TXLF-10-L-R-S

0.5 mL thin wall PCR tubes

PCR-05-C

Invitrogen

Qubit Assay Tubes

Q32856

BIOSHARP

Metal Block-

AxygenChoose either one of the listed brands (with * mark).

1.5. Practice Tips
Stereo-seq Chip T Slide
Includes 4 Stereo-seq Chip T Slides containing one Chip T (1cm*1cm) on each slide.
Stereo-seq Chip P Slides and Stereo-seq Chip T Slides are differentiated by a laserengraved label at the end of the slide.

Chip T

Chip T

Chip T

Chip T

Stereo-seq Chip T Slide Storage
Always store unused slides in their original slide container and keep them sealed with asealable aluminum bag at -25°C ~ 8°C. Keep sealed with tape or another re-sealable bag.
Always KEEP the desiccant in the bag.

9

1. INTRODUCTION

STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

Stereo-seq Slide Cassette
STOmics Stereo-seq Accessory Kit contains a Stereo-seq Cassette and removable
Gaskets which need to be assembled prior to use.

For a demonstration video of Stereo-seq Slide Cassette assembling and removal, please refer tothe link or by scanning the QR code:
https://en.stomics.tech/resources/videos/list.html

Stereo-seq Slide Cassette Assembly
Pick up the Stereo-seq Slide Cassetteand flip it over. Insert the gasket into theStereo-seq Slide Cassette, ensuring thatthe cutouts are aligned.

a.

Take the Stereo-seq Slide Cassetteand gasket out of the STOmics Stereo-seq
Accessory Kit.

b.

Press the gasket down to secure it in
the cassette.

d.

c.

10

Use a power dust remover to blow off
any debris on the gasket if necessary.

1. INTRODUCTION

STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

Pick up the Stereo-seq Chip Slideand flip it over with the chip surface
facing down. Align the engraved labelwith the long edge of the Stereo-seq Slide
Cassette.

e.

Ensure that the chips are aligned within the empty space of the gasket and avoid
touching the chip surface with the gasket or cassette during slide placement. Insert theStereo-seq Chip Slide under the bottom 4 tabs.

f.

Support the back of the cassette withboth middle fingers. Place your left thumbbetween tab 1 and tab 2, and place yourright thumb between tab 3 and tab 4.

g.

11

Press the upper side (A side) of theslide (near the edge) evenly and thensimultaneously press the top edge down
firmly with both index fingers to clip theslide in place until you hear it click.

h.

1. INTRODUCTION

STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

Press along both edges of the Stereoseq Slide Cassette to ensure that theStereo-seq Chip Slide is locked in place.

i.

Look closely at the Stereo-seq Slide Cassette and confirm that the slide is clipped inplace.

j.

Stereo-seq Slide Cassette Removal
Flip the cassette over and firmly press the upper side down to release the slidefrom the tabs, while gently supporting the back of the Stereo-seq Chip Slide with boththumbs to prevent the Stereo-seq Chip Slide from falling off.

a.

b.

Lift the Stereo-seq Chip Slide from the engraved label end.

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1. INTRODUCTION

STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

1.6. Precautions and Warning•This product is intended for research use only, not for use in diagnostic procedures.
Read all instructions in this manual carefully before using the product.

•

Before performing experiments with the kits, users are recommended that you
ensure that they are familiar with related instruments, and operate them according
to manufacturer’s instructions.

•

Instructions provided in this manual are intended for general use only, andoptimization may be required for specific applications.

•Thaw the reagents in the kits properly prior to use. For enzymes, centrifuge briefly
and keep them on ice until further use. For other reagents, thaw them first atroom temperature followed by inverting several times to mix them properly, andcentrifuge them briefly before placing on ice for further use.

•mRNA capture will be compromised or absent for any scratched areas on the front
surface of the chip.

•

To prevent cross-contamination, we recommend using filtered pipette tips. Use a
new tip each time for pipetting different solutions.

•

We recommend using a thermal cycler with heated lids for PCR reactions. Unlessotherwise stated, pre-heat the thermal cycler to reaction temperature before use.

•

Improper handling of samples and reagents may contribute to aerosol
contamination of PCR products, resulting in data inaccuracy. Therefore, we
recommend two distinctly separated working areas in the laboratory for PCR
reaction preparation and PCR product cleanup tests. Use designated pipettes
and equipments for each area and execute regular cleaning (with 0.5% sodiumhydrochloride or 10% bleach) to ensure a clean and sterile working environment.

•

Do not consume any sample or reagent, and avoid direct contact of reagents withskin and eyes. In case of an accident, immediately wash the affected area thoroughlywith a large amount of water. Seek emergency medical assistance if needed.

13

1. INTRODUCTION

CHAPTER 2
SAMPLE AND EXPERIMENTPREPARATIONSTEREO-CITE PROTEO-TRANSCRIPTOMICS SET User ManualFor guidelines on sample embedding, sectioning, and mounting, please refer to Sample
Preparation, Sectioning, and Mounting Guide for Fresh Frozen Samples on Stereo-seq
Chip Slides (Document No.: STUM-SP001).

15

2. SAMPLE AND EXPERIMENTAL PREPARATIONCHAPTER 3
PILOT STEREO-CITE
EXPERIMENTSTEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

TotalSeq™-A antibodies from Biolegend are developed for single-cell CITE-seq
technology and may not be suitable for all tissue sections. For users selecting theirantibodies, it is recommended to conduct an initial immunofluorescence (IF) teston slides with the tissue type of interest to ensure that the selected TotalSeq™-A
antibodies are well suited for the tissue and an optimal dilution ratio of the antibodies
can be determined. Once the concentration of each antibody is determined, theformal Stereo-CITE experiment can be performed.
This chapter can be skipped if TotalSeq™-A Mouse Universal Cocktail, V1.0
(Biolegend, Cat. No. 199901) or TotalSeq™-A Human Universal Cocktail, V1.0
(Biolegend, Cat. No. 399907) are being used. Please directly proceed to Chapter
4, the standard operating procedure for the Stereo-CITE workflow, and use therecommended dilution ratios following the instructions from Biolegend.

3.1. Experiment Preparation
Unless otherwise specified, nuclease-free water is used for all reagents being
prepared prior to this experiment.

Reagent

Preparation Steps

Maintenance

4% PFAMix well after thawing and aliquot to a 2
mL/tube for storage. Equibrate to room
temperature before use.

-20°C for no morethan 1 month

5X SSC

Dilute 5 mL of 20X SSC to 20 mL.

Room Temperature

0.1X SSC

Dilute 100 µL of 20X SSC to 20 mL;
dilute 250 µL of 20x SSC to 50 mL.

Room Temperature

Filtered Serum
BufferTake the serum out in advance and thaw
it. Mix horse serum and goat serum in a 1:1
ratio. Filter the mixture using a 0.22 μm filter
membrane (compatible with a syringe-stylesterile disposable syringe). Recommend
aliquoting 200 μL per tube (for one round of
permeabilization and proteo-transcriptomics -20°C
workflow). Before the experiment, thaw thealiquoted serum at -20°C. Centrifuge at 4°C,
14000g for 10 min and keep it for later use.
Approximately 20 μL of serum is needed for
one chip, and any remaining serum can bereused.

Do not freeze and thaw the aliquot more than 3 times.
Salmon SpermDNA

Thaw on ice before use, 20µL/chip.

17

-20°C

3. PILOT STEREO-CITE EXPERIMENTSTEREO-CITE PROTEO-TRANSCRIPTOMICS SET User ManualPrimary &Secondary
Antibody

Thaw on ice following the original productinstructions, centrifuge for 10 min at 14000g.
4°C or -20°C
The Primary Abs should include the isotype
Abs

Dilute theantibodies if
needed.

Dilute the antibodies if needed.

Place on ice until
ready to use

Dilute 100% Triton X-100 into 10% Triton
10% Triton X-100 X-100 with Nuclease-free water if there is no
ready-to-use 10% Triton X-100

Room Temperature

FcR Blocking
Reagent

For mouse samples, use TruStain Fcx™ PLUS
(CD16/32) Antibody; for human samples, use 4°C
TruStain Fcx™

GlycerolTake it out in advance and equilibrate to
Room Temperatureroom temperature at least 5 min prior to use.

Decrosslinking
Reagent

Take it out in advance and equilibrate to
Room Temperatureroom temperature at least 5 min prior to use.

Other Preparation
Equipments

Set up

Note

Cryostat

Set the cryostat chamber temperature to-20°C and specimen disc temperature(object temperature) to -10°C ~ -15°C.

The specimen
disc temperaturedepends on thetissue type.

Centrifuge

Set the centrifuge to cool to 4°C in advance -PCR Thermal Cycler

Check if there is any
abnormality with
Set the PCR module to 37°C (heating lid at
the PCR Thermal
42°C)
Cycler and replace
it if necessary.

Fluorescence
Microscope

Ensure that the microscope is equippedwith at least DAPI, FITC, TRITC, CY5

18

Please select thechannels according
to the fluorescentemission
wavelengths ofyour secondary
antibodies.

3. PILOT STEREO-CITE EXPERIMENTSTEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

3.2. Cryosection Preparationa.

Set PCR thermal cycler to 37°C with heated lid set to 42°C in advance with a PCR
adaptor.

b.

Set cryostat chamber temperature to -20°C and specimen disc temperature (object
temperature) to -10°C~-15°C.
If the specimen disc is over-cooled, it could lead to tissue section cracking duringsectioning, while sections would wrinkle when the disc temperature is too high.
Optimal specimen disc temperature depends on the tissue type.

c.

Place forceps, brushes, and razor blades inside the chamber for pre-cooling.

d. Take the OCT-embedded tissue sample out of the -80°C freezer and place it in

the cryostat chamber for 30 min to allow it to equilibrate to cryostat chamber
temperature.

e.

During temperature equilibration, prepare the blocking buffer (2 sections for every
antibody), please refer to Table 3-1 and put it on ice.
Table 3-1 Blocking Buffer

Components

1X ( µL)

2X + 10% ( µL)

3X + 10% ( µL)

4X + 10% ( µL)

5X SSC

120

252

372

492

10% Triton X-100

2

4.2

6.2

8.2

Salmon Sperm DNA,
20
sheared

42

62

82

FcR Blocking
Reagent*

10

21

31

41

Filtered Serum

20

42

62

82

Nuclease-free water 28

58.8

86.8

114.8

Total

420

620

820

200

FcR Blocking Reagent is for blocking of Fc receptors on the cell membrane.
Please select either one according to the host species of your sample. For humantissue, please use Catalog# 422031 and for mouse tissue, please use Catalog#
156604.
1X blocking buffer is enough for blocking and preparation of Primary antibody
incubation buffer of one tissue section.
f.

Remove the sample outer covers (aluminum foil) and trim the embedded tissue
block to the appropriate size (sectioning area smaller than 0.9 cm x 0.9 cm).

g.

By using OCT, mount the embedded tissue block onto the specimen disc/holder ofthe cryostat chamber.

h.

Do a final trimming if necessary to ensure a good fit between the tissue section and
Stereo-seq Chip T. Now, the specimen is ready for cryosection.
19
3. PILOT STEREO-CITE EXPERIMENTSTEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

3.3. Tissue Mounting on Microscopic Slidesa.

Add over 1/3 Volume of 4% PFA to the slide container and ensure that the volume
can cover the tissue on the chip and slide. Prepare enough 4% PFA in a 50 mL
corning tube or an empty slide container at a volume that could submerge the entire
microscopic slide (about 1/3 of the container).

b.

Tissue mounting: 1 section per 1 slide isrecommended (2 sections per antibody, as 1 section
is for the negative control).

c.

Tissue mounting can be achieved via either the cold
method (option A) or warm method (option B).

1/3 ofthe totalvolumeA. Cold Method
1) Place microscope slide inside the chamber with the front-side facing up andpre-cool inside the cryostat chamber for 1~6 min.
Prolonged cooling for over 6 min may cause mist formation on the chip surface.
2) Perform cryosection, then carefully flatten the tissue section out by gently
touching the surrounding OCT with cryostat brushes. Place a tissue section onto
the center of the slide carefully with forceps and brushes. Ensure that the tissue
section is complete and without wrinkles.
3) Immediately pick up the microscope slide and place a finger on the backsideof the microscope slide directly under the tissue for a few seconds to allow thesection to adhere to the surface.
4) Once all tissue mounting is complete, immediately dry the microscope slide at
37°C on a PCR thermal cycler with PCR adaptor for 5 min.
When performing cold mounting, mind the timeinterval between each tissue
section placement. Longer time intervals (>5 min)could result in tissue wrinkle formation.

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3. PILOT STEREO-CITE EXPERIMENTSTEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

B. Warm Method
1) Perform cryosection and carefully flatten
the tissue sections out by gently touching thesurrounding OCT with cryostat brushes.
2) Move the tissue section to the edge, flip the
microscope slide and aim the tissue section onto
the center of the slide by gently touching thesection with the microscope slide.
3) Check and see if the tissue section has been
mounted on to the microscope slide.
4) Turn the microscope slide over, and immediately
dry it at 37°C on a PCR thermal cycler with PCR
adaptor front-side up for 5 min.

3.4. Tissue Fixationa.

After drying, immediately immerse the tissue-mounted microscope slide in 4% PFA
solution at room temperature and fix for 10 min.

b.

After fixation, move the 4% PFA container to a fume hood. Meanwhile, prepare a
50mL centrifuge tube filled with at least 30mL of 0.1X SSC.

c.

Take the microscope slide out of the 4% PFA solution and immediately immerse theslide in a 50 mL centrifuge tube with at least 30 mL of 0.1X SSC for 10 sec.

d. Take out the slide and immediately wipe off excess SSC solution from around andthe back of the slide with Kimwipes without touching the tissue to ensure that there
is no residual liquid surrounding the tissue.

e.

Use a Super PAP Pen (hydrophobic barrier pen) on the microscope slide to draw
a circle around the tissue on the slide, creating a hydrophobic isolation zone thatprevents subsequent additional fluid outflow.
Subsequent fluid changes are carried out in the hydrophobic isolation area.

f.

Transfer the processed slides to an immunohistochemistry wet cassette.
All the liquid must be added after the hydrophobic isolation residue dries.

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3. PILOT STEREO-CITE EXPERIMENTSTEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

3.5. Tissue Blocking and Primary Antibody Incubationa.

Vortex the blocking solution that was prepared in Table 3-1 and add no more than
100 μL/section of blocking solution dropwise on the tissue surface within thehydrophobic zone. Incubate for 20 min at room temperature.
The amount of blocking solution used per section is dependent on the sizeof the hydrophobic area. For a hydrophobic area size of 0.5cm × 0.5cm, therecommended blocking solution volume is 30 μL/section.

hydrophobic
circleDuring incubation, prepare the Primary Antibody Incubation Buffer according toTable 3-2. Vortex the buffer and leave it on ice until use.

b.

Table 3-2 Primary Antibody Incubation Buffer
Components

1X ( µL)

Primary Antibody

X

Blocking Buffer

100-X

Total

100

The Biolegend TotalSeq™-A can be diluted at a ratio of 1:250 for the initialattempt. If the fluorescent staining does not meet the requirements, it isrecommended to titrate the antibody specifically to determine the final antibody
dilution ratio by referring to Appendix C.
c.

Aspirate the blocking solution, slowly add the primary antibody incubation solution(no primary antibody in the negative control group) with no more than 100 μL/
section, and incubate the primary antibody for 45 min at room temperature.
Avoid tissue drying, as it may lead to the generation of non-specific signals thatinterfere with the final results.

d. During primary antibody incubation, prepare the secondary antibody incubation

solution detailed in Table 3-3 according to the optimal dilution ratio of the secondary
antibody. After vortex mixing and brief centrifugation, leave the secondary antibody
solution on ice in the dark until use.

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Table 3-3 Secondary Antibody Incubation Buffer
Components

1X ( µL)

5X SSC

60

Secondary Antibody

X

Nuclease- free water

40-X

Total

100

We recommend using Alexa Fluor Plus Series Fluorescent Secondary Antibodies
at a 1:500 dilution concentration. If fluorescent secondary antibodies from othervendors are used, please adjust the dilution ratio according to the manufacturer's
instructions.

3.6. Secondary Antibody Incubationa.

Remove the primary antibody incubation solution of the experimental group andthe blocking solution of the negative control group.

b.

Add 100 μL 0.1X SSC per section. Incubate at room temperature for 1 min and thenremove.
Avoid tissue drying, as it may lead to the generation of non-specific signals thatinterfere with the final results.

c.

Repeat step b. once.

d. Slowly add no more than 100 μL secondary antibody incubation solution per section

from the non-tissue area in both the experimental group and the negative control
group. Incubate for 15 min at room temperature in the dark.

e.

Remove the secondary antibody incubation solution.

f.

Add 100 μL 0.1X SSC per section. Incubate at room temperature for 1 min and thenremove.

g.

Repeat step f. once.

h.

Hold on to the slide with one hand and completely dry the tissue using a power dust
remover at a distance 2-3 cm away from the tissue surface by blowing gently fromone side at a 30-degree angle horizontal to the plane of the slide.

23

3. PILOT STEREO-CITE EXPERIMENTSTEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manuali.

Pipette 5 µL glycerol gently onto the center of the tissue on each slide without
introducing air bubbles.

Glycerolj.

With a pair of forceps, place one end of the coverslip onto the tissue edge while
holding the other end and then gradually lower the coverslip onto the tissue.
Ensure that the tissue is completely covered by glycerol and the coverslip. To avoid
fluorescent bleaching, IMMEDIATELY proceed to imaging.

Coverslip

Coverslip

Ensure that the coverslip is clean without any dust or debris. Wiping with an
alcohol swab or blowing with a power dust remover could be used for cleaning.

3.7. Imaging
a.

Fluorescent images are taken using a fluorescence microscope with an image
stitching function, and the fluorescence configuration is recommended as follows:•

Light source wavelength range: 380-680 nm•

Monochrome camera (≥ 12 bit)•

DAPI filter cube (ref. Excitation 375/28 nm, Emission 460/50 nm)•

FITC filter cube (Reference: Excitation 480/30 nm, Emission 525/50 nm)•

TRITC filter cube (ref. Excitation 545/25 nm, Emission 605/70 nm)•

CY5 filter cube (Reference: Excitation 620/50 nm, Emission 690/50 nm)
24

3. PILOT STEREO-CITE EXPERIMENTSTEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual•

Maximum pixel size: 5 μm/pixel•

Exposure time: 1 ms~2 s (depending on the characteristics of the antibody used)b.

Transfer the slides to the microscope stage and remove the light shield.

c.

c. Select the epifluorescence scanning mode, adjust the fluorescence channel
according to the fluorescence excitation light of the secondary antibody used, andscan using 10X objective. Following scanning, save both the FOV images and stitched
images.

3.8. Stereo-CITE Pilot Experiment Results
As shown in Figure 2, taking the negative control Ctrl and CD68 staining of mouse
thymus tissue as an example, it is confirmed that using the primary antibody at the ratioof 1:250 yielded the desired staining results. If you do not get the expected results, it will
be necessary to determine the optimal antibody dilution by antibody titration.

Figure 1 Immunofluorescent staining of the control and CD68 in mouse thymus

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3. PILOT STEREO-CITE EXPERIMENTCHAPTER 4
Stereo-CITE PROTEOTRANSCRIPTOMICS SETSTANDARD OPERATINGPROCEDURE

STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

4.1. Experimental Preparation
Unless otherwise specified, nuclease-free water is used for all reagents being
prepared prior to this experiment.

Reagent

Preparation Steps

Maintenance

4% PFAMix well after thawing and aliquot to
2mL/tube for storage. Equibrate to room
temperature before use.

-20°C for no morethan 1 month

5X SSC

Dilute 5 mL of 20x SSC to 20 mL

Room Temperature

0.1X SSC

Dilute 100 μL of 20x SSC to 20 mL; Dilute 250
μL of 20x SSC to 50 mL

Room Temperature

Wash Buffer

Add 160µL RI into 3040 µL of 0.1X SSC for one
Room Temperature
tissue section

Filtered Serum
BufferTake the serum out in advance and thaw
it. Mix horse serum and goat serum in a 1:1
ratio. Filter the mixture using a 0.22 μm filter
membrane (compatible with a syringe-stylesterile disposable syringe). Recommend
aliquoting 200 μL per tube (for one round of
permeabilization and proteo-transcriptomics -20°C
workflow). Before the experiment, thaw thealiquoted serum at -20°C. Centrifuge at 4°C,
14000g for 10 min and keep it for later use.
Approximately 20 μL of serum is needed for
one chip, and any remaining serum can bereused.

Do not freeze and thaw the aliquot more than 3 times.
Salmon SpermDNA

Thaw on ice before use, 20µL/chip-20°C

Blocking Reagent Thaw on ice before use, 15 µL/chip

-20°C

RI

Place on ice until
ready to use

Thaw on ice before use, 210 µL/chip

50X Diluted DAPI Dilute DAPI with 5X SSC at the ratio of 1:50
Primary &Secondary
Antibody

4°C in the dark for 1
day

Thaw on ice following the original productinstructions, centrifuge for 10 min at 14000g.
4°C or -20°C
The Primary Abs should include the isotype
Abs
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4. STANDARD OPERATING PROCEDURE

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Diluted Primary& Secondary(Optional)

Dilute the antibodies if needed.

Dilute 100% Triton X-100 into 10% Triton
10% Triton X-100 X-100 with Nuclease-free water if there is no
ready-to-use 10% Triton X-100

Place on ice until
ready to use
Room Temperature

FcR Blocking
Reagent

For mouse samples, use TruStain Fcx™ PLUS
(CD16/32) Antibody; for human samples, use 4°C
TruStain Fcx™

GlycerolTake it out in advance and equilibrate to
Room Temperatureroom temperature at least 5 min prior to use.

Decrosslinking
Reagent

Take it out in advance and equilibrate to
Room Temperatureroom temperature at least 5 min prior to use.
a. Equilibrate the lyophilized powder tubes
at room temperature for 5 min;b. Place the lyophilized powder tube in an
empty 2mL EP tube at 10,000 g for 30 S atroom temperature;

TotalSeq™-A
Mouse Universal c. Add 27.5 μL of 1XPBS and incubate for 5
min at room temperature;
Cocktail, V1.0
Or
d. Vortex and centrifuge at 10,000 g for 30 sec 4°C
TotalSeq™-A
at room temperature;
Human Universal
e. Transfer the entire volume (27.5 μL) of the
Cocktail, V1.0
reconstituted antibody Cocktail solution to a
(Optional)
new EP tube and centrifuge at 14,000 g for 10
min at 4 °C;
f. Aspirate 25 μL of the supernatant, transfer
to a new EP tube, and place on ice for later
use.
Room temperaturefor 48 hr (Storing
Prepare at least 2mL of 0.01N HCL per
longer than 48 hr will0.01N HCl
sample. Configure HCL to 0.01N. Measureaffect the desired pH.
and ensure the pH = 2.
Use within 48 hr ofpreparation.)
Always use freshly prepared 0.01N HCl (pH = 2.0 ± 0.1). For pre-made 0.1N HCL andnewly purchased HCL, check the pH prior to conducting experiments.
10X
Permeabilization
Reagent Stock
Solution

Add 1 mL of freshly prepared 0.01N HCl todissolve PR Enzyme (red cap, in powderform), and thoroughly mix the reagent by
pipetting.

-20°C

Do not vortex the permeabilization enzyme. Mix by pipetting before use. Aliquot
this 10X stock solution to prevent freeze-thaw cycles.
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4. STANDARD OPERATING PROCEDURE

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1X
Make 1X PR solution (150 μL / chip) by
Permeabilization diluting 10X PR stock solution with 0.01N
Reagent Solution HCl.
Short spin the primer tube, dissolve RT Oligo
in 79 μL TE buffer. Close the lid, vortex the
RT Oligotube for 15 sec at highest speed and shortspin the tube.

Place on ice until
ready to use. Can beon ice for up to 6 hr.
-20°C

Aliquot the unused RT Oligo to avoid freeze-thaw cycles and store at -80°C.
PR Rinse Buffer

Take it out in advance and equilibrate to
Room Temperatureroom temperature at least 5 min prior to use.

PR Rinse Buffer(with 5% RI)

Prepare at least 100 μL per chip (95 μL PR
Rinse Buffer with 5 μL RI)

Place on ice until
ready to use

Other Preparation
Equipment

Set up

Note

Cryostat

Set the cryostat chamber temperature to-20°C and specimen disc temperature(object temperature) to -10°C ~ -15°C.

The specimen
disc temperaturedepends on thetissue type.

Metal Bath

70°C for decrosslinking
37°C for pre-heating the permeabilization
enzyme-Set the temperature in the following order:
Check the PCR
37°C for slide drying and permeabilization
Thermal Cycler for(heating lid at 42°C); 42°C for reverse
PCR Thermal Cycler
abnormalities. If
transcription (heating lid at 47°C); 55°C fornecessary, replacetissue removal and cDNA release (heating lid
it.
at 60°C).

Fluorescence
Microscope

Please select thechannels according
to the fluorescent
Ensure that the microscope is equipped withemission
at least DAPI, FITC, TRITC, CY5
wavelengths ofyour secondary
antibodies.

Centrifuge

4°C is used forcentrifugation ofserum, primaryCentrifuge cocktail lyophilized powder at
antibodies,room temperature, then adjust the centrifuge
secondarytemperature to 4 °C in advance.
antibodies, andreconstituted
antibody cocktails.
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4. STANDARD OPERATING PROCEDURE

STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User ManualFor guidelines on sample embedding, sectioning, and mounting, please refer toSample Preparation, Sectioning, and Mounting Guide for Fresh Frozen Samples
on Stereo-seq Chip Slides (Document No.: STUM-SP001).
During the temperature equilibration step at cryosection preparation, prepare theblocking buffer by referring to Table 4-1 and put it on ice. Meanwhile, take enough
4% PFA solution (at least 400 μL 4% PFA solution per chip) from 4°C to equilibrate
to room temperature.
Table 4-1 Blocking Buffer
Components

1X ( µL)

2X + 10% ( µL)

3X + 10% ( µL)

4X + 10% ( µL)

5X SSC

180

378

558

738

10% Triton X-100

3

6.3

9.3

12.3

Blocking Reagent

15

31.5

46.5

61.5

Salmon Sperm DNA,
30
sheared

63

93

123

FcR Blocking
Reagent*

15

31.5

46.5

61.5

RI

15

31.5

46.5

61.5

Filtered Serum

30

63

93

123

Nuclease-free water 12

25.2

37.2

49.2

Total

630

930

1230

300

FcR Blocking Reagent is for blocking of Fc receptors on the cell membrane. Please
select either one according to the host species of your sample. For human tissue,
please use Catalog# 422031 and for mouse tissue, please use Catalog# 156604.
1X blocking buffer is enough for blocking and preparation of Primary antibody
incubation buffer of one tissue section.

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4.2. Tissue Fixationa.

Assemble the Cassette and Gasket then place the Stereo-seq Chip Slide in the
Cassette according to guidance written in 1.5 Practice Tips. It is recommended topractice with a regular blank glass slide.
Do not to touch the front-side of the chip while assembling the Stereo-seq Slide
Cassette.

b.

Place the Stereo-seq Slide Cassette in a fume hood, and add 400 μL of 4% PFA
solution per well. Seal the cassette with sealing tape, and incubate for 10 min atroom temperature.

c.

After fixation, remove the sealing tape, and tilt the Stereo-seq Slide Cassette slightly
at an angle less than 20°. Remove the 4% PFA solution from a corner of the wellusing a pipette. Keep the chip surface moist.

d. Immediately add Wash Buffer (400 μL per well) and incubate at room temperaturefor 1 min.

e.

Tilt the Stereo-seq Slide Cassette slightly at an angle less than 20°, and remove theWash Buffer solution from the corner of the well using a pipette. Keeping the chipand tissue surface moist.
Avoid drying out the tissue during the liquid exchange process. Ensure that that
all the tissue sections are completely submerged.

f.

Repeat wash steps d. and e. one more time.

4.3. Tissue Blocking and Primary Antibody Incubationa.

Blocking: Transfer the Stereo-seq Slide Cassette out of the fume hood to a bench
top and add 150 μL blocking buffer per chip onto the tissue surface immediately.
Incubate the chip at room temperature for 20 min.

b.

While waiting, prepare the primary antibody incubation solution according to Table
4-2 with the actual number of actual number of individual TotalSeq™-A antibodies
used. After vortex mixing and brief centrifugation, leave the primary antibody
incubation solution on ice until use. For TotalSeq-A™ Mouse Universal Cocktail,V1.0 (Biolegend, Cat. No. 199901) and TotalSeq-A™ Human Universal Cocktail, V1.0
(Biolegend, Cat. 399907), please refer to Table 4-3 for preparing the primary antibody
incubation solution.

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Table 4-2 Primary Antibody Incubation Solution(For freely combined individual TotalSeq™-A antibodies of your interest)
Components

1X ( µL)

Blocking Buffer150-(V1+V2+...+Vn)

Primary Antibody #1

V1

Primary Antibody #2

V2

...

...

Primary Antibody #N

Vn

Total

150

Antibody 1~N should contain the corresponding isotype control antibody. Ensurethat to centrifuge the TotalSeq™-A antibody before use, and try to avoid the tip of
the pipette coming in direct contact with the bottom of the tube.
Table 4-3 Primary Antibody Incubation Solution(For TotalSeq™-A Cocktails)
Components

1X ( µL)

Blocking Buffer

137.5

Universal Cocktail

12.5

Total

150

When working with human samples including tonsil, lymph node, esophagus, lung,intestines, and cervix, or mouse samples including spleen, thymus, and liver, it isrecommended to use 12.5 μL of human or mouse TotalSeq-A universal cocktails per
chip. However, for first-time users, it is suggested to use 25 μL per chip instead toensure an accurate and reliable result.
c.

Pipette to remove the blocking buffer from one corner of the well. Ensure the chipand tissue surface remain moist.
Avoid drying out the tissue during the liquid exchange process. It is recommended
to perform the procedure one chip at a time.

d. Slowly add 150 μL of the primary antibody incubation solution per well onto thechip by first pipetting one droplet at each corner of the chip and then adding therest of the solution to the middle to merge all the droplets. Incubate for 45 min atroom temperature.

e.

During primary antibody incubation, prepare the secondary antibody incubation
solution according to Table 4-4. Vortex the mixture and leave it on ice in the dark
until use.

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Table 4-4 Secondary Antibody Incubation Solution
Components

1X ( µL)

5X SSC

90

RI

7.5

Secondary Antibody

0.3

Nuclease-free water

52.2

Total

150

The solution should be stored in the dark. We recommend using Alexa Fluor Plus
Series Fluorescent Secondary Antibodies at a 1:500 dilution concentration. If
fluorescent secondary antibodies from other vendors are used, please adjust thedilution ratio according to the manufacturer's instructions.
f.

Pipette to remove the primary antibody incubation solution from one corner of thewell. Ensure the chip and tissue surface remain moist.

g.

Add 200 μL Wash Buffer per well and incubate the chip at room temperature for 1
min.

h.

Tilt the Stereo-seq Slide Cassette slightly at an angle of less than 20°, remove theWash Buffer from the corner of the well using a pipette, and keep the chip and tissue
surface moist.

i.

Repeat wash steps f. and g. one more time.
Avoid drying out the tissue during the liquid exchange process. It is recommended
to perform the step one chip at a time.

j.

Slowly add 150 μL secondary antibody incubation solution per well onto the chip by
first pipetting one droplet at each corner of the chip and then adding the rest of thesolution to the middle to merge all the droplets., and incubate in the dark at room
temperature for 15 min.

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4.4. DAPI Staininga.

During the secondary antibody incubation, prepare DAPI staining solution according
to Table 4-5. Vortex and centrifuge briefly then leave it on ice in the dark until use.
Table 4-5 DAPI Staining Solution

Components

1X ( µL)

5X SSC90

50X-diluted DAPI solution

1.5

RI

7.5

Nuclease-free water

51

Total

150

b.

Pipette to remove the secondary incubation solution from one corner of the well,and keep the chip and tissue surface moist.

c.

Add 200 μL Wash Buffer per well and incubate the chip at room temperature for 1
min.

d. Tilt the Stereo-seq Slide Cassette slightly at an angle less than 20°, remove the Wash

Buffer from one corner of the well using a pipette, and keep the chip and tissue
surface moist.

e.

Repeat step c. and d. once to achieve 2 washes.
Avoid drying out the tissue during the liquid exchange process. It is recommended
to perform the step one chip at a time.

f.

Slowly add 150 μL DAPI Staining Solution per well onto the chip by first pipetting
one droplet at each corner of the chip and then adding the rest of the solution to the
middle to merge all the droplets. Incubate in the dark at room temperature for 2
min.

g.

Pipette to remove the solution from one corner of the well. Keep the chip and tissue
surface moist.

h.

Add 200 μL Wash Buffer per well and incubate the chip at room temperature for 1
min.

i.

Tilt the Stereo-seq Slide Cassette slightly at an angle of less than 20°, remove theWash Buffer from the corner of the well using a pipette, and keep the chip and tissue
surface moist.

j.

Repeat wash steps h. and i. one more time.

k.

Remove the slide from the Stereo-seq Slide Cassette according to instructions in 1.5
Practice Tips.

l.

Hold on to the slide with one hand and completely dry the tissue further with apower dust remover in the other hand at a distance 2-3 cm away from the tissue
surface by blowing gently from one side at a 30-degree angle horizontal to the planeof the slide.
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STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

Alternatively, centrifuge the Stereo-seq Chip Slide for 10 sec in a slide spinner tocompletely dry the chips (Labnet Slide Spinner, C1303-T ).
Ensure no residual solution is left on the chip.
m. Pipette 5 μL glycerol gently onto the center of the tissue on each chip without

introducing air bubbles.

Glyceroln.

With a pair of forceps, place one end of the coverslip onto the chip while holding
the other end and then gradually lower the coverslip onto the chips. Ensure thatthe chips are completely covered by glycerol and the coverslip. To avoid fluorescentbleaching, IMMEDIATELY proceed to imaging.

Coverslip

Coverslip

Ensure that the coverslip is clean without any dust or debris. Wiping with an
alcohol swab or blowing with a power dust remover could be used for cleaning.

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STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

4.5. Imaging
a.

b.

Fluorescence images are taken using a fluorescence microscope with image stitching
functions, and the fluorescence configuration is recommended as following:•

Light source wavelength range: 380-680 nm•

Monochrome camera (≥ 12 bit)•

DAPI filter cube (ref. Excitation 375/28 nm, Emission 460/50 nm)•

FITC filter cube (Reference: Excitation 480/30 nm, Emission 525/50 nm)•

TRITC filter cube (ref. Excitation 545/25 nm, Emission 605/70 nm)•

CY5 filter cube (Reference: Excitation 620/50 nm, Emission 690/50 nm)•

Maximum pixel size: 5 μm/pixel•

Exposure time: 1 ms~2 s (depending on the characteristics of the antibody used)

Create a new folder in a fluorescent microscope-connected PC, name it with the chip
ID number and other essential information.
Note: Only use letters, numbers, and underscores in folder naming. Special
characters and spacings are not allowed.
Example: Y00035N1

c.

Take fluorescence images from the chip with the following microscope setting: DAPI
channel, 4X and 10X objective lenses, with stitching function.

d. Add a few drops of water (~2μL) to the microscope loading platform, then place theStereo-seq Chip Slide on the water to better adhere to the platform and avoid thedisplacement of the Stereo-seq Chip Slide during image scanning.

e.

Remove the light shield, switch to 4X objective lens, select the DAPI channel andselect the chip area of interest. Adjust Brightness and Gain. The specific parameters
will vary according to different microscopes, as long as the tissue can be imaged
clearly. But the light intensity should be kept in the lower range to prevent
fluorescence quenching.

f.

After scanning with 4X objective lens, take fluorescence images from the chip withthe following microscope setting: DAPI channel, 10X objective lens, full scan on
capture area.

As shown in Figure 4, taking the DAPI channel image of mouse thymus tissue as an
example, the red box in the middle picture is the selected tissue area, and the small bluebox is the added focal points. The picture on the left shows a screenshot of a focal field
window selected outside the tissue, which should ensure that the tracklines are clear
and distinct. The picture on the right is a screenshot of a focal field window selected in
the tissue area, which should ensure that the tissue outline and contour is clear.

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Figure 2 DAPI staining of mouse braing.

Once you complete the first scan of the DAPI channel, switch directly to the next
fluorescent channel (FITC channel, TRITC channel, or CY5 channel) depending on thesecondary antibody selected WITHOUT moving the Stereo-seq Chip Slide, rescanning the map, re-adding the focal points, or changing the red box of the selected
tissue area. Save original tile (FOV) image files and stitched images. Simply create a
new folder, name and save it with the chip number_IF format name (e.g., Y00035N1_
IF) in the computer connected to the fluorescence microscope. Then adjust the focus
and exposure until the stained tissue is clearly displayed. Finally, complete the fullscan on the capturing area with a 10X objective lens.

h.

Based on the results of the immunofluorescence staining image, whether the
TotalSeq™-A primary antibody has been successfully binded to the antigens can be
determined. The judging criteria are dependent on the type of antibody added, andthe images obtained should be consistent with prior knowledge of
immunofluorescent staining. For example, taking Figure 4 as an example, due to theaddition of multiple primary antibodies, all areas of the thymus tissue have shown afluorescent signal (Figure 4, left image), while the cell membrane can be identified
when zoomed into a specific cell membrane primary antibody (Figure 4, rightimage). The cell membrane staining can be clearly visualized after magnification,
indicating that there is no occurrence of unwanted antibody binding.

Figure 3 IF staining of the mouse thymus

The staining of the secondary antibody can be used to determine whether the
primary antibody has been successfully binded to the antigens. If unsuccessful, it is
not recommended to continue with subsequent experiments.
37

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STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manuali.

Save original tile (FOV) images files and stitched images.

j.

j. Open the ImageStudio software and the Image Quality Control functional module
within the software. Upload your nuclei-stained (DAPI) image then run Image QC
according to the ImageStudio User Manual within the software.
The captured nuclei-stained image needs to pass ImageQC in order to proceed tofurther image analysis (image “register”) in Stereo-seq Analysis Workflow (SAW)
pipelines.
If Image QC failed, continue with the experimental procedures and later perform
optimal image analysis under the guidance of your local Field Application Scientist.

k.

After imaging, remove the Stereo-seq Chip Slide from the microscope platform andkeep the coverslip mounted for the next steps.
For mouse liver samples, please skip Section 4.6 and proceed directly from Section
4.7.

4.6. Tissue Decrosslinkinga.

Set aside the Decrosslinking Reagent you prepared in [Experimental Preparation].

b.

Place the PCR adaptor into the PCR thermal cycler and set one PCR module to 70°C
with the lid set to 75°C and another PCR module to 37°C with the lid set to 42°Cc.

Warm up the Decrosslinking reagent to 70°C for at least 10 min.

d. Grip onto the coverslip with a pair of forceps and pull it to slide over the Stereo-seq

chip Slide slowly until the chips and the coverslip are fully separated.

Coverslip

Coverslipe.

Wipe off the excess solution from around and the back of the slide with Kimwipes
without touching the chips. Ensure that there is no liquid residue around the chip.

f.

Assemble the Cassette and a new Gasket then place the Stereo-seq chip Slide in the
Cassette according to guidance written in 1.5 Practice Tips. It is recommended topractice with a regular blank glass slide.

g.

Add 400 μL Wash Buffer per well and incubate the chip at room temperature for 1
min.

h.

Tilt the Stereo-seq Slide Cassette slightly at an angle less than 20°, remove the Wash
Buffer from the corner of the well using a pipette, and keep the chip and tissue
surface moist.
38

4. STANDARD OPERATING PROCEDURE

STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manuali.

Repeat Step h. and i. once.

j.

Place the PCR adaptor in the PCR thermal cycler first. Ensure that your PCR thermal
cycler has been switched on and set to 70°C with the lid set to 75°C. Place the Stereoseq Slide Cassette on the PCR adaptor and add 400 µL Decrosslinking Buffer per well.
Seal the cassette with new sealing tape. Close the heated lid and incubate at 70°C for
15 min.

k.

While waiting, pre-warm the 1X Permeabilization Reagent Solution you prepared inExperimental Preparation to 37°C in advance (no more than 30 min).

l.

Transfer the Stereo-seq Slide Cassette to the bench and gently remove the sealing
tape. Do not remove the decrosslinking solution. Equilibrate the cassette to room
temperature for 10 min.

4.7. Tissue Permeabilizationa.

Set aside the 2 mL of HCl and 1X Permeabilization Reagent Solution you prepared inExperimental Preparation.

b.

Ensure that your PCR thermal cycler has been switched on and set to 37°C and theheating lid has been set to 42°C. Place the PCR adaptor in the PCR thermal cycler,then close the heated lid to pre-heat the adaptor for 3 min.
Keep the Stereo-seq Chip Slide on the same PCR thermal cycler for the Reverse
Transcription step.

Temperature

Time

Cycle

(Heated lid) 42°C

on

-

37°C

60 min

1

37°C

hold

-

c.

Thaw RT Reagent, RT Additive and RT Oligo on ice.

d. Once the cassette equilibrates to room temperature, tilt the Stereo-seq Slide

Cassette slightly at an angle of less than 20°, remove the Decrosslinking Solution
from a corner of the well using a pipette, and keep the chip and tissue surface moist.

e.

Add 400 µL Wash Buffer per well.

f.

Tilt the Stereo-seq Slide Cassette slightly at an angle of less than 20°, remove theWash Buffer from a corner of the well using a pipette.

g.

Add 150 µL 1X Permeabilization Reagent Solution per chip onto the chip by first
pipetting one droplet at each corner of the chip and then adding the rest of thesolution to the middle to merge all the droplets.
Ensure that the chip is completely covered with 1X Permeabilization Reagent
Solution.

h.

Seal the Stereo-seq Slide Cassette with a new sealing tape and place it on the PCR
Adaptor. Close the hot lid and let the chip incubate inside the PCR thermal cycler at
37°C with the optimal permeabilization time.
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Optimal permeabilization time is pre-determined by Stereo-seq Permeabilization
Kit (111KP118). For more information, refer to the user manual of Stereo-seq
Permeabilization Set for Stereo-CITE Proteo-Transcriptomics Application User
Manual.
While waiting for permeabilization to be done, prepare RT mix according to Table 4-6
and leave it on ice until use.

i.

Table 4-6 RT Mix
Components

1X ( µL)

2X + 10% ( µL)

3X + 10% ( µL)

4X + 10% ( µL)

RT Reagent

160

336

496

656

RT Additive

10

21

31

41

RI

10

21

31

41

RT Oligo

10

21

31

41

ReverseT Enzyme

10

21

31

41

Total

200

420

620

820

j.

Keep the Stereo-seq Slide Cassette in the PCR thermal cycler and remove the sealing
tape. Tilt the cassette slightly at an angle of less than 20°. Pipette to remove the 1x
Permeabilization Reagent Solution from a corner of the well, and avoid touching thechip surface.

k.

Add 200 µL PR Rinse Buffer (with 5%RI) per well.

l.

Tilt the Stereo-seq Slide Cassette slightly at an angle of less than 20°, remove the PR
Rinse Buffer from the corner of the well using a pipette, and keep the chip and tissue
surface moist.
Ensure that to not dry the chip completely.

m. Continue with reverse transcription immediately to avoid RNA degradation.

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4.8. Reverse Transcriptiona.

Set the temperature of the same PCR Thermal Cycler and PCR Adaptor that wereused in the Tissue Permeabilization step to 42°C with the heated lid set to 47°C.

b.

Pipette up and down, then short spin the prepared RT Mix. Gently add 200 μL of RT
Mix per chip along the side of each well, ensuring that the well surface is uniformly
covered with RT Mix.

c.

Apply sealing tape to Stereo-seq Slide Cassette and ensure that it is sealed tightly.
Incubate the Stereo-seq Slide Cassette at 42°C for 3 hr or longer (no longer than 16hr) with the following incubation protocol.

Temperature

Time

Cycle

(Heated lid) 47°C

on

-

42°C

3 hr - 16 hr

1

42°C

hold

-

4.9. Tissue Removal
PrepareReagent

Preparation Steps

MaintenanceTR buffer

Heat the buffer for 5 min at 55 °C to dissolve
the precipitate. Equilibrate it to room
Room Temperaturetemperature prior to use.

cDNA Release
BufferHeat the buffer for 5 min at 55 °C to dissolve
the precipitate. Equilibrate it to room
Room Temperaturetemperature prior to use.

If white precipitate is observed in the buffer, dissolve them by heating the bufferat 55 °C again and equilibrate to room temperature before mixing.
a. Check and ensure that the PCR Thermal Cycler with PCR Adaptor has been set to
55°C and the heated lid has been set to 60°C.
b. Remove the Stereo-seq Slide Cassette from the 42°C PCR Adaptor and then remove
the sealing tape.
When removing the sealing tape, hold on to the Stereo-seq Slide Cassette with
one hand without applying forces to Side A and Side B of the cassette in order toprevent the Stereo-seq Chip Slide from falling off of the cassette.
41

4. STANDARD OPERATING PROCEDURE

STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manualc. Slightly tilt the Stereo-seq Cassette, remove RT Mix with a pipette from the corner of
each well without touching the chip surface.
d. Add 400 µL TR Buffer into each well and incubate the Stereo-seq Slide Cassette at
55°C on the PCR Adaptor for 20 min with the following incubation protocol.

Temperature

Time

Cycle

(Heated lid) 60°C

on

-

55°C

20 min

1

55°C

Hold

-

e. While waiting, prepare cDNA Release Mix according to Table 4-7.
Table 4-7 cDNA Release Mix
2X + 10% ( µL)

3X + 10% ( µL)

4X + 10% ( µL)

cDNA Release Buffer 380

836

1254

1672

cDNA Release
Enzyme

20

44

66

88

Total

400

880

1320

1760

Components

1X ( µL)

f. Slightly tilt the Stereo-seq Slide Cassette, remove TR Buffer with a pipette from thecorner of each well without touching the chip surface.
If tissue removal is not complete, add 400 μL of 0.1X SSC and pipette up and down
the well to remove the remaining tissue on the chip. Slightly tilt the Stereo-seq
Slide Cassette, remove 0.1X SSC solution from the corner of each well without
touching the chip surface.

4.10. cDNA & ADT Release and Collection
a. Add 400 µL of cDNA Release Mix per chip prepared in 4.8-e into each well of theStereo-seq Slide Cassette.
b. Apply sealing tape to Stereo-seq Slide Cassette and ensure that it is sealed tightly.
Incubate the Stereo-seq Slide Cassette at 55°C for 3 hr or longer (no longer than 18 hr)
with the following incubation protocol.

Temperature

Time

Cycle

(Heated lid) 60°C

on

-

55°C

3 hr - 18 hr

1

55°C

Hold

-

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STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

Stop Point:
DNA collection step may be left overnight. If it is left overnight, ensure that the Stereoseq Slide Cassette has been sealed tightly with the sealing tape.
c. After the reaction, completely collect the cDNA Release Mix from each well into a new
1.5 mL tube.
d. Add 100 µL of nuclease-free water per chip into each well. Pipette up and down towash the chip surface thoroughly and then collect it into the same 1.5 mL tube with thecDNA Release Mix.
Ensure that to collect as completely as possible to retrieve enough cDNA on thechip. cDNA Release Mix should be about 400 μL after incubation (the volume might
be less than 400 μL). It is required to combine the collected cDNA Release Mix withthe 100 μL nuclease-free water before proceeding to the next step.
Ensure all the chip ID numbers on the Slide have been recorded, as it is required fordownstream analysis.

4.11. cDNA Purification and Amplification
Background Information
For bead-based purification, we recommend using DNA Cleanup Beads AMPure®
XP(Agencourt, Cat. No. A63882), SPRIselect (Beckman Coulter, B23317/B23318/B23319)
or VAHTS™ DNA Clean Beads (Vazyme, Cat. No. N411-02). If magnetic beads from othersources are used, please optimize the cleanup conditions before getting started.

Before Use•

To ensure the DNA capture efficiency of the magnetic beads, equilibrate the beads toroom temperature 30 min before use.

•

Vortex or pipette up and down to ensure the beads are thoroughly mixed every time
before use.

•

The volume of magnetic beads directly affects the distribution of purified DNA
fragments.

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Operation Notes
1. In the magnetic separation step, please allow the solution to become completely
clear before removing the supernatant. This process usually takes approximately
2-3 min, but can be longer or shorter depending on the type of magnetic separation
rack being used.

2. When collecting the supernatant with a pipette after magnetic separation, avoid
taking up the beads. Instead of collecting the entire supernatant fraction, leave 2-3µL in the tube to avoid the pipette from directly contacting the beads. If the beads
are mistakenly taken up, dispense everything and redo the magnetic separation.

3. Use freshly prepared 80% ethanol (at room temperature) to wash the beads. Keepthe sample tube on the magnetic separation rack during the washing step. Do notshake or disturb the beads.

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4. After the 2nd wash of beads with ethanol, try to remove all the liquid within the
tube. You may centrifuge briefly to accumulate any remaining liquid at the bottomof the tube, then separate beads magnetically, and remove the remaining liquid byusing a small volume pipette.

5. After washing twice with ethanol, air-dry the beads at room temperature. Drying
usually takes approximately 5-10 min depending on the lab temperature andhumidity. Watch closely until the pellet appears sufficiently dry with a matteappearance, then continue to the elution step with TE Buffer.

Sufficient drying

Over dried with cracked surface

6. During the elution step, do not touch the beads with the pipette tip when removing
the supernatant. Contamination of a DNA sample with beads may affect subsequent
purification steps. Therefore, to avoid the pipette tip from directly contacting thebeads, always collect the eluate in 2 µL less than the initial volume of the eluant
used for the elution.

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STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

7. Pay attention when opening/closing the lid of a sample tube on a separation
rack. Strong vibrations may cause samples or beads to spill from the tubes.
Hold the body of the tube while opening the lid.

a.

Equilibrate the magnetic beads to room temperature for at least 30 min.

b.

cDNA Purification Procedures with 1X Magnetic Bead
1) Mix the collected cDNA (450-490 µL) with the beads in a ratio of 1 : 1. Vortex the mix
then incubate it at room temperature for 10 min.
2) Spin down and place the tube onto a magnetic separation rack for 3 min until theliquid becomes clear.
3) Carefully transfer the supernatant with a pipette to a new 2.0 mL tube. The
supernatant contains ADT products and can be stored at room temperature
temporarily. Refer to 4.11 for ADT product purification).
4) Keep the tube on the magnetic separation rack and add 1 mL of freshly prepared
80% ethanol to wash the beads by rotating the tube on the magnetic rack. Incubate
for 30 sec and carefully remove and discard the supernatant.
Always place the pipette tips on the tube wall and away from the magnetic beads.
Do not disturb the beads while transferring the supernatant (If foam is seen on thecap, clean the cap with 80% ethanol).
5) Repeat step 4) one more time.
6) Keep the tube on the magnetic rack, and open the lid to air-dry the beads at room
temperature until no wetness (reflectiveness) is observed. Drying times will vary but
will be approximately 5-8 min.
7) Add 22 µL of nuclease-free water to the dried beads. Mix the beads and nucleasefree water by vortexing. Incubate at room temperature for 5 min. Spin down briefly
and place the sample tube onto a magnetic separation rack for 3-5 min until theliquid becomes clear.
8) Transfer the supernatant (21 µL) into a new 0.2 mL PCR tube.
9) Add another 22 µL of nuclease-free water to the dried beads in step 7) for a second
elution. Mix the beads and nuclease-free water by vortexing. Incubate at room
temperature for 5 min. Spin down briefly and place the sample tube onto a magnetic
separation rack for 3-5 min until the liquid becomes clear.
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10) Transfer the supernatant (21 µL) into the 0.2 mL PCR tube in step 8) and obtain a
combined eluted cDNA (~42 µL)
If collected eluted cDNA is less than 42 μL, simply top it up with nuclease-free water.

c.

Resuspend the beads with 42 µL of nuclease-free after cDNA collection. Store
resuspended beads at 4°C until the cDNA final product has passed QC.
d. Prepare PCR Mix by referring to Table 4-8. The total volume for the PCR reaction is

100 μL.

Table 4-8 PCR Mix
Components

1X ( µL)

2X + 10% ( µL)

3X + 10% ( µL)

4X + 10% ( µL)

cDNA Amplification
Mix

50

110

165

220

cDNA Primer

8

17.6

26.4

35.2

Eluted cDNA

42

2 x 423 x 424 x 42Total

100

2 x 100

3 x 100

4 x 100e.

Mix gently and short spin before placing the reaction tube in a thermal cycler.
Amplify the eluted cDNA based on the PCR program stated in Table 4-9.
Table 4-9 PCR Program for Amplification (for 100 µL)

Temperature

Time

Cycle

(Heated lid) 105°C

on

-

95°C

5 min

1

98°C

20 sec58°C

20 sec

72°C

3 min

72°C

5 min

1

12°C

Hold

-

15

While waiting, refer to section 4.11 for purification and amplification of ADT
product.
f.

Prepare Qubit dsDNA Mix and record the concentration of PCR product according toTable 4-10.

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Table 4-10 Qubit dsDNA Mix
Components

1X ( µL)

Invitrogen™ Qubit dsDNA HS Buffer

198

Qubit dsDNA HS Reagent 200X

1PCR Product

1

Total

200

g.

Vortex the mix and then take 1 µL of the PCR product and measure its concentration
using Qubit dsDNA HS Kit. The DNA concentration is usually more than 5 ng/µL.
For troubleshooting purposes, we recommend leaving about 2 µL of the PCR
product in a PCR tube.

h.

Use magnetic beads to purify the PCR product in a volume ratio of 1 : 1 (DNA :beads).
1) Mix the cDNA PCR product (100 μL) with beads in a ratio of 1 : 1. Vortex the mixture
then incubates it at room temperature for 10 min.
2) Spin down and place the sample tube onto a magnetic separation rack for 3 min
until the liquid becomes clear. Carefully remove and discard the supernatant with apipette.
3) Keep the tube on the magnetic separation rack and add 200 μL of freshly
prepared 80% ethanol to wash the beads by rotating the tube on the magnetic
separation rack. Incubate for 30 sec then carefully remove and discard thesupernatant.
Always place the pipette tips on the tube wall and away from the magnetic
beads. Do not disturb the beads while transferring the supernatant.
4) Repeat step 3) one more time.
5) Keep the tube on the magnetic rack, and open the lid to air-dry the beads at room
temperature until no wetness (reflectiveness) is observed. Drying times will vary but
will be approximately 5-8 min.
6) Vortex the dried beads with 42 μL of TE buffer. Incubate at room temperaturefor 5 min. Spin down briefly and place the sample tube onto a magnetic rack for
3-5 min until the liquid becomes clear. Transfer 40 μL supernatant to a new 1.5 mL
centrifuge tube.

Stop Point:
The purified cDNA sample can be stored at −20°C for up to 1 month.

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For troubleshooting purposes, we recommend storing the beads with 40 µL of
nuclease-free water at 4°C after purification till your cDNA final product has
passed QC.
i.

Take 1 μL of the cDNA sample and measure and record the concentration of thepurified cDNA with Qubit dsDNA HS Kit.

j.

k. Analyze the sample (dilution might be required) on an Agilent Bioanalyzer HighSensitivity chip or other library quality control platform such as Tapestation (Agilent
Technologies), LabChip® GX, GXII, GX Touch (PerkinElmer) and Fragment Analyzer™
(Advanced Analytical).
A qualified cDNA sample should have a main fragment distribution peak
appearing at around 500-1,200 bp (Figure 4), and a yield higher than 20 ng.

Figure 4. A representative Agilent Bioanalyzer 2100 analysis result of a PCR amplified cDNA sample

4.12. ADT Product Purificationa.

Equilibrate the magnetic beads to room temperature for at least 30 min. Prepare
80% ethanol freshly and equilibrate to room temperature before use.

b.

ADT Product Purification Procedures with 2.0X Magnetic Bead:
1) Mix the collected supernatant in the 2.0 mL tube collected in section 4.10 b-3)
with 500 μL beads. Vortex the mix then incubate it at room temperature for 10 min.
2) Spin down and place the tube onto a magnetic separation rack for 5 min until theliquid becomes clear.
3) Carefully remove and discard the supernatant with a pipette (If foams are seen on
the cap, discard them with a pipette).

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4) Keep the 2.0 mL tube on the magnetic separation rack and add 1.5 mL of freshly
prepared 80% ethanol to wash the beads by rotating the tube on the magnetic rack.
Incubate for 30 sec and carefully remove and discard the supernatant.
Always place the pipette tips on the tube wall and away from the magnetic
beads. Do not disturb the beads while transferring the supernatant (If foams
are seen on the cap, clean the cap with 80% ethanol).
5) Repeat step 4) one more time.
6) Keep the tube on the magnetic rack, and open the lid to air-dry the beads atroom temperature until no wetness (reflectiveness) is observed. Drying times willvary but will be approximately 5-8 min.
7) Add 22 μL of nuclease-free water to the dried beads. Mix the beads and nucleasefree water by vortexing. Incubate at room temperature for 5 min. Spin down briefly
and place the sample tube onto a magnetic separation rack for 3-5 min until theliquid becomes clear.
8) Transfer the supernatant (~21 µL) into a new 0.2 mL PCR tube.
9) Add another 22 μL of nuclease-free water to the dried beads. Mix the beads andnuclease-free water by vortexing. Incubate at room temperature for 5 min. Spindown briefly and place the sample tube onto a magnetic separation rack for 3-5 min
until the liquid becomes clear.
10) Transfer the supernatant (~21 µL) into the 0.2 mL PCR tube in step 9) and obtaina combined eluted ADT product.
If the collected eluted ADT is less than 42 μL, simply top it up with nuclease-freewater.

c.

Store the beads with 40 μL of nuclease-free water at 4°C after collecting the eluted
cDNA till your cDNA final product has passed QC.
d. Prepare PCR Mix by referring to Table 4-11. The total volume for the PCR reaction is

100 μL.

Table 4-11 ADT PCR Mix
2X + 10% ( µL)

3X + 10% ( µL)

4X + 10% ( µL)

ADT Amplification Mix 50

110

165

220

ADT Primer

8

17.6

26.4

35.2

Eluted ADT42

2 x 423 x 424 x 42Total

100

2 x 100

3 x 100

4 x 100

Componentse.

1X ( µL)

Mix gently and short spin before placing the reaction tube in a thermal cycler.
Amplify the eluted ADT product based on the PCR program stated in Table 4-12.

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Table 4-12 PCR Program for Amplification (for 100 µL)

Temperature

Time

Cycle

(Heated lid) 105°C

on

-

95°C

5 min

1

98°C

20 sec58°C

20 sec

72°C

1 min

72°C

5 min

1

12°C

Hold

-

18

While waiting, refer to section 4.11 for purification and amplification of ADT
product.
Prepare Qubit dsDNA Mix and record the concentration of PCR product according toTable 4-13.

f.

Table 4-13 Qubit dsDNA Mix
Components

1X ( µL)

Invitrogen™ Qubit dsDNA HS Buffer

198

Qubit dsDNA HS Reagent 200X

1PCR Product

1

Total

200

g.

Vortex the mix and then take 1 µL of the PCR product and measure its concentration
using Qubit dsDNA HS Kit. The DNA concentration is usually more than 5 ng/µL.
For troubleshooting purposes, we recommend leaving about 2 µL of the PCR
product in a PCR tube.

h.

ADT PCR product Purification Procedures with 2.0X Magnetic Bead.
1) Mix the ADT PCR product (100 μL) with beads in a ratio of 1 : 2. Vortex the mixture
then incubates it at room temperature for 10 min.
2) Spin down and place the sample tube onto a magnetic separation rack for 5 min
until the liquid becomes clear. Carefully remove and discard the supernatant with apipette.
3) Keep the tube on the magnetic separation rack and add 300 μL of freshly prepared
80% ethanol to wash the beads by rotating the tube on the magnetic separation rack.
Incubate for 30 sec then carefully remove and discard the supernatant.
Always place the pipette tips on the tube wall and away from the magnetic
beads. Do not disturb the beads while transferring the supernatant.

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4) Repeat step 3) one more time.
5) Keep the tube on the magnetic rack, and open the lid to air-dry the beads at room
temperature until no wetness (reflectiveness) is observed. Drying times will vary but
will be approximately 5-8 min.
6) Vortex the dried beads with 42 μL of TE buffer. Incubate at room temperaturefor 5 min. Spin down briefly and place the sample tube onto a magnetic rack for
3-5 min until the liquid becomes clear. Transfer 40 μL supernatant to a new 1.5 mL
centrifuge tube.

Stop Point:
The purified ADT PCR product sample can be stored at −20°C for up to 1 month.
For troubleshooting purposes, we recommend storing the beads with 40 μL of
nuclease-free water at 4°C after purification till your ADT PCR final product has
passed QC.
i.

Take 1 μL of the ADT PCR product sample, measure and record the concentration of
the purified ADT PCR product with Qubit dsDNA HS Kit.

j.

Analyze the sample (dilution might be required) on an Agilent Bioanalyzer HighSensitivity chip or other library quality control platform such as Tapestation (Agilent
Technologies), LabChip® GX, GXII, GX Touch (PerkinElmer) and Fragment Analyzer™
(Advanced Analytical).
A qualified ADT PCR product sample should have a main fragment distribution
peak appearing at around 170- 220 bp (Figure 5), and the concentration is above
10 ng/µL.

Figure 5. A representative Agilent Bioanalyzer 2100 analysis results in a PCR amplified ADT product52

4. STANDARD OPERATING PROCEDURE

CHAPTER 5
LIBRARY PREPARATIONSTEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

The later steps will require the Stereo-seq Library Preparation kit. Please ensure thatyou have purchased the kit separately and prepare the reagents in advance prior to
library construction.
Table 5-1 Library Preparation Kit

Stereo-seq Library Prep Kit Cat. No.: 111KL114
Component

Reagent Cat. No.

TME

1000028515(white)4 µL

×1

Stop Buffer

1000028516(white)

40 µL

×1

TMB

1000028517(white)40 µL

×1

PCR Barcode Primer
Mix (Barcode 57-64)

1000028519

50 µL

×1

PCR Barcode Primer
Mix (Barcode 81-88)

1000029088

50 µL

×1

PCR Barcode Primer
Mix (Barcode 89-96)

1000029089

50 µL

×1

PCR Barcode Primer
Mix (Barcode 97-104)

1000029180

50 µL

×1

PCR Amplification Mix

1000029181

400 µL × 1

Storage Temperature:-25°C~-18°C

Cap Color

Transported bycold chain

Quantity (tube)

Expiration Date:refer to label

Please ensure that a substantial amount of dry ice remains with the kits upon
arrival.
Performance of products may only be guaranteed before their expiration date.
Proper performance is also subject to the products being transported, stored, andused in appropriate conditions.

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Table 5-2 Barcode Amplification Kit

Stereo-seq 16 Barcode Amplification Kit Cat. No.: 111KB016
Component

Reagent Cat. No.

PCR Barcode Primer Mix 1

1000043201

25 µL

×1

PCR Barcode Primer Mix 2

1000043202

25 µL

×1

PCR Barcode Primer Mix 3

1000043203

25 µL

×1

PCR Barcode Primer Mix 4

1000043204

25 µL

×1

PCR Barcode Primer Mix 5

1000043205

25 µL

×1

PCR Barcode Primer Mix 6

1000043206

25 µL

×1

PCR Barcode Primer Mix 7

1000043207

25 µL

×1

PCR Barcode Primer Mix 8

1000043208

25 µL

×1

PCR Barcode Primer Mix 91000043209

25 µL

×1

PCR Barcode Primer Mix 101000043210

25 µL

×1

PCR Barcode Primer Mix 111000043211

25 µL

×1

PCR Barcode Primer Mix 121000043212

25 µL

×1

PCR Barcode Primer Mix 13

1000043213

25 µL

×1

PCR Barcode Primer Mix 14

1000043214

25 µL

×1

PCR Barcode Primer Mix 15

1000043215

25 µL

×1

PCR Barcode Primer Mix 16

1000043216

25 µL

×1

PCR Amplification Mix

1000043217

25 µL

×1

Storage Temperature:-25°C~-18°C

Cap Color

Transported bycold chain

Quantity (tube)

Expiration Date:refer to label

This kit does not contain TME, Stop Buffer, TMB, etc. For fresh frozen samples,
please use the Stereo-seq Library Preparation Kit (4 RXN, Cat. No.: 111KL114).

55

5. LIBRARY PREPARATIONSTEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

5.1. Experimental Preparation
Unless otherwise specified, nuclease-free water is used for all reagents being
prepared prior to this experiment.

Reagent

Preparation Steps

Maintenance

80% Ethanol

Dilute 100% ethanol to 80%

Roomtemperature up to
1 day

Magnetic beads

Take it out in advance and equilibrate toroom temperature at least 30 min prior to
use.

4°C

10-fold diluted TME

Dilute 1 µL of TME to 10 µL with TE buffer

On ice up to 1 hr

DO NOT dilute all the TME at once. Volume provided should be enough for 4
dilutions.
Stop Buffer

Take it out in advance and equilibrate toroom temperature at least 30 min prior to
use

Room
temperature up to
1 day

5.2. cDNA Fragmentation and Amplificationa.

Use 20 ng cDNA sample prepared in section 4.10. for the following fragmentation
reaction.

Prepare the Fragmentation Reaction Mix on ice through gentle pipetting according
to Table 5-3. Pipette 10-fold diluted TME up and down before mixing with the rest.
After a short spin, gently mix the solution through pipetting while keeping the tube
on ice.
Avoid vortexing TME.

b.

Table 5-3 Fragmentation Reaction Mix
Components

1X ( µL)

TMB

410-fold diluted TME

1cDNA Product

X

Nuclease-free water

15-X

Total

20

cDNA Input: X (µL) = 20 ng/Concentration of cDNA (ng/µL)
56

5. LIBRARY PREPARATIONSTEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

Program a thermocycler according to Table 5-4. When the module starts to heat up,
put the reaction tube into the thermocycler.

c.

Please DO NOT leave the reaction tube on ice after taking it out of the thermocycler.
Table 5-4 Fragmentation Reaction Program

Temperature

Time(Heated lid) 60°C

on

55°C

10 min12°C

Hold

d. After the fragmentation reaction program is done, take out the reaction tube, shortspin it to collect all the liquid to the bottom and then leave it at room temperature.
Add 5 µL of Stop Buffer to the fragmentation reaction mix to terminate thefragmentation process, and then pipette to mix thoroughly. Incubate the mix at
room temperature for 5 min.
Set up PCR Library Mix according to Table 5-5 to start the amplification process offragmented cDNA.

e.

Table 5-5 PCR Library Mix
Components

1X ( µL)

Fragmentation product25

PCR Barcode Primer Mix

25

PCR Amplification Mix

50

Total

100

Selection principle of PCR Barcode Primer Mix:f.

•

1-4 Libraries per sequencing run, choose the kit with Cat. No.: 111KL114 andrefer to Appendix A for guidelines for using PCR Barcode Primer Mix;•

4 < libraries <16 per sequencing run, choose the kit with Cat. No.: 111KB016 andrefer to Appendix B for guidelines for using PCR Barcode Primer Mix;
Vortex and spin down briefly the reaction mix prepared above. Incubate it in athermocycler with the following incubation protocol (Table 5-6) and start the
program.

57

5. LIBRARY PREPARATIONSTEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

Table 5-6 PCR Amplification Program (for 100µL)

Temperature

Time

Cycle

(Heated lid) 105 °C

on

-

95°C

5 min

1

98°C

20 sec58°C

20 sec72°C

30 sec

72°C

5 min

1

12°C

Hold

-

g.

13

Take 1 μL of the PCR product and use the Qubit dsDNA HS Kit to measure theconcentration. The concentration is usually around 10-100 ng/μL.

5.3. PCR Product Size Selectiona.

Mix the PCR product obtained above with the magnetic beads in a volume ratio of 1
: 0.55 (PCR product : beads = 100 µL : 55 µL) in a PCR tube. Vortex the mixture thenincubate it at room temperature for 5 min.

b.

Short spin the reaction mix and place the tube onto a magnetic separation rack for
3 min until it becomes clear. Then, carefully transfer the supernatant to a new PCR
tube.
Keep the supernatant and discard the beads.

c.

Add 15 µL of beads to the new PCR tube with the supernatant from step b. Vortex tomix thoroughly. Incubate at room temperature for 5 min.

d. Spin down and place the tube onto a magnetic separation rack for 3-5 min until it

becomes clear. Carefully discard the supernatant with a pipette.

e.

Keep the tube on the magnetic separation rack and add 200 µL of freshly prepared
80% ethanol to wash the beads by rotating the tube on the magnetic rack. Incubate
for 30 sec and carefully remove the supernatant with a pipette.

f.

Repeat step e. one more time.

g.

Spin-down the tube and put it on the magnetic rack to collect beads on the sidewall.
Use a smaller pipette tip to remove the remaining liquid and discard it.

h.

Air-dry the beads for 3-5 min until the bead surface is not reflective. Do not allow thebeads to overdry and crack.
58

5. LIBRARY PREPARATIONSTEREO-CITE PROTEO-TRANSCRIPTOMICS SET User ManualMix the dried beads with 20 µL of TE buffer, vortex to mix and incubate at room
temperature for 5 min. Spin down briefly and place the centrifuge tube onto amagnetic separation rack for 3 min until the liquid becomes clear. Transfer thesupernatant to a new 1.5 mL tube.

i.

Keep the supernatant.
Take 1 μL of purified PCR product and measure the concentration with Qubit dsDNA
HS Kit. Use Bioanalyzer, Tapestation (Agilent Technologies), LabChip® GX, GXII, GX
Touch (PerkinElmer), Fragment Analyzer™ Advanced Analytical) or other equipment
based on the principle of electrophoretic separation to detect the fragment
distribution of the purified PCR products. The main peak of fragment distribution isrequired to be 200-600 bp (Figure 6). Normally, the PCR yield is higher than 100 ng.

j.

Figure 6. Agilent 2100 Bioanalyzer fragment size distribution of the purified PCR product5.4. ADT Product Amplification
It is required to use different sample barcodes when sequencing ADT Libraries andtranscriptome libraries together. Please refer to Appendix A and Appendix B for
details of the usage rules.
If not specified, all the reagents should use Nuclease-free water for dilution.
a.

Use 20 ng ADT product prepared in section 4.11. for the following fragmentation
reaction.

b.

Set up PCR Library Mix according to Table 5-5 to start the amplification process of
ADT product.

59

5. LIBRARY PREPARATIONSTEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

Table 5-7 ADT Product PCR Library Mix
Components

1X ( µL)

ADT product (20ng)

X

Nuclease-free water

25 - X

PCR Barcode Primer Mix

25

PCR Amplification Mix

50

Total

100

Selection principle of PCR Barcode Primer Mix:c.

•

1-4 Libraries per sequencing run, choose the kit with Cat. No.: 111KL114 andrefer to Appendix A for guidelines for using PCR Barcode Primer Mix;•

4 < libraries <16 per sequencing run, choose the kit with Cat. No.: 111KB016 andrefer to Appendix B for guidelines for using PCR Barcode Primer Mix;
Vortex and spin down briefly the reaction mix prepared above. Incubate it in athermocycler with the following incubation protocol (Table 5-6) and start the
program.
Table 5-8 PCR Amplification Program (for 100µL)

Temperature

Time

Cycle

(Heated lid) 105 °C

on

-

95°C

5 min

1

98°C

20 sec58°C

20 sec

72°C

1 min

72°C

5 min

1

12°C

Hold

-

8

d. Take 1 μL of the PCR product and use the Qubit dsDNA HS Kit to measure theconcentration. The concentration is usually around 10-100 ng/μL.

60

5. LIBRARY PREPARATIONSTEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

5.5. ADT PCR Product Size Selectiona.

Mix the PCR product obtained above with the magnetic beads in a volume ratio of
1 : 2 (PCR product : beads = 100 μL : 200 μL) in a PCR tube. Vortex the mixture thenincubates it at room temperature for 10 min.

b.

Short spin the reaction mix and place the tube onto a magnetic separation rack for
3-5 min until it becomes clear. Then discard the supernantant.
Keep the beads and discard the supernantant.

c.

Keep the tube on the magnetic separation rack and add 400 μL of freshly prepared
80% ethanol to wash the beads by rotating the tube on the magnetic rack. Incubate
for 30 sec and carefully remove the supernatant with a pipette.

d. Repeat step c. one more time.
e.

Spin-down the tube and put it on the magnetic rack to extract the beads out of theliquid. Use a smaller pipette tip to remove the remaining liquid and discard it.

f.

Air-dry the beads for 5-8 min until the bead surface is not reflective or cracked.

g.

Mix the dried beads with 24 μL of TE buffer, vortex to mix and incubate at room
temperature for 5 min. Spin down briefly and place the centrifuge tube onto amagnetic separation rack for 3-5 min until the liquid becomes clear. Transfer thesupernatant to a new 1.5 mL tube.
Keep the supernatant.

h.

Take 1 μL of purified PCR product and measure the concentration with Qubit dsDNA
HS Kit. Use Bioanalyzer, Tapestation (Agilent Technologies), LabChip® GX, GXII, GX
Touch (PerkinElmer), Fragment Analyzer™ (Advanced Analytical) or other equipment
based on the principle of electrophoretic separation to detect the fragment
distribution of the purified PCR products. The main peak of fragment distribution isrequired to be 200-250 bp (Figure 7). Normally the PCR yield is higher than 100 ng.

Figure 7. Agilent 2100 Bioanalyzer fragment size distribution of the ADT PCR product61

5. LIBRARY PREPARATIONCHAPTER 6
LIBRARY CONSTRUCT& SEQUENCING

STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User ManualThis chapter introduces the compatible sequencing instruments and sequencing
reagents for the Stereo-seq system.
Please refer to the user manual of High-throughput Sequencing Primer Kit (STOmics),
Cat. no. 940-000037-00 for DNB preparation.
Sequencing strategy for Stereo-CITE transcriptome and ADT libraries:•

When sequencing on DNBSEQ-T7RS:·Pooling both Stereo-CITE transcriptome and ADT libraries on the same flowcell isrecommended.
·It is recommended to pool DNB libraries (DNB pooling) when sequencing both
Stereo-CITE transcriptome and ADT libraries.
·For base balancing, it is recommended to mix the protein ADT and transcriptome
libraries with different sample barcodes at a 1:1 input mass ratio, or with lessprotein ADT library.

•

The cDNA and ADT library structure when pooled on the same T7 flowcell areillustrated in Figure 8 and Figure 9.

Use the following parameters to perform the sequencing run:
• Choose paired-ended mode with 50 cycles of Read 1 and 100 cycles of Read 2 and an
additional 10 cycles of sample barcode. Use dark cycles on Read 1 from 26 to 40 cycles.

Figure 8. Stereo-CITE Transcriptome Library Structure

Figure 9. Stereo-CITE ADT Library Structure (when pooled with cDNA library together on T7)

Please read the corresponding user manual High-throughput Sequencing Primer Kit(STOmics), Cat. no. 940-000037-00 carefully before performing sequencing and strictly
follow the instructions. If you have any questions about sequencing, please contact yourlocal technical support for DNBSEQ platforms.
63

6. LIBRARY CONSTRUCT & SEQUENCING

STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User ManualThis chapter introduces the compatible sequencing instruments and sequencing
reagents for the Stereo-seq system.
Sequencing strategy for Stereo-CITE transcriptome and ADT libraries:•

When sequencing on DNBSEQ-G400:·Pooling both Stereo-CITE transcriptome and ADT libraries on the same flowcell is
not recommended.
·It is recommended to sequence Stereo-CITE transcriptome and ADT libraries
separately on different flowcells.

Use the following parameters to perform the sequencing run:•cDNA library:·Choose paired-ended mode with 50 cycles of Read 1 and 100 cycles of Read 2 andan additional 10 cycles of sample barcode. Use dark cycles on Read 1 from 26 to 40
cycles.

Figure 10. Stereo-CITE Transcriptome Library Structure•

Protein ADT library:·Choose paired-ended mode with 50 cycles of Read 1 and 36 cycles of Read 2 andan additional 10 cycles of sample barcode. Use dark cycles on Read 1 from 26 to 40
cycles and on Read 2 from 1 to 21 cycles.

Figure 11. Stereo-CITE ADT Library Structure (when sequenced individually on G400)

Please read the corresponding user manual High-throughput Sequencing Primer Kit(STOmics), Cat. no. 940-000037-00 carefully before performing sequencing and strictly
follow the instructions. If you have any questions about sequencing, please contact yourlocal technical support for DNBSEQ platforms.
64

6. LIBRARY CONSTRUCT & SEQUENCING

STEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

Appendix A: PCR Barcode Primer Mix Use Rules
The PCR Barcode Primer Mix in this kit is a pre-mixed barcode combination with abalanced set of bases, which can be randomly selected for use by the customer. Splitting
barcode is required in sequencing for two or more samples in the same lane (in order todistinguish your sequencing samples). If there is only one sample, do not split barcode
for sequencing. The following table is the barcode sequence number in each pre-set PCR
Barcode Primer Mix.
PCR Barcode Primer
Mix Name

Contains the Barcode Sequence Number

PCR Barcode Primer Mix(Barcode 57~64)

57

58

59

60

61

62

63

64

PCR Barcode Primer Mix(Barcode 81~88)

81

82

83

84

85

86

87

88

PCR Barcode Primer Mix(Barcode 89~96)

89

90

91

92

93

94

95

96

PCR Barcode Primer Mix(Barcode 97~104)

97

98

99

100

101

102

103

104

65

APPENDIXSTEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

Appendix B: 16 PCR Barcode Primer Mix Use Rules
The Stereo-CITE 16 Barcode Amplification Kit provides 16 types of PCR Barcode Primer
Mix. It is designed for a large number of samples batched library construction and multisample mixed sequencing. This kit is based on the design principle of base balance, andhas been tested by repeated experiments. The best Barcode combinations have been
selected for use with the Stereo-CITE Library Preparation Kit. To ensure the best results,
please read the following usage rules carefully.
Any sample with the same barcode cannot be sequenced in the same lane.
Based on the base balancing principle, the PCR Barcode Primer Mix should be used ingroups with the following grouping rules:
4 PCR Barcode Primer Mixes in groups: 1~4, 5~8, 9~12, 13~16, a total of 4 sets.
Ensure that to centrifuge the PCR Barcode Primer Mix thoroughly before use. Gently
open the lid during use, to prevent the liquid from splashing and avoid crosscontamination. Pipetting to mix well and shortly centrifuge while using. Close thelid in time after use.
N types of PCR Barcode Primer Mix Method: take the same volume of each; mix
them and add the mixture to the sample.

When the data amount requirements for each sample are the same:
For different sample sizes, please refer to the recommended Barcode combinations
shown below:
Sample/lane Method 1

Method 2

Method 3

Method 4

1

1 to 45 to 89 to 1213 to 162

Sample 1: 1 and 2
Sample 2: 2 and 4

Sample 1: 5 and 6
Sample 2: 7 and 8

Sample 1: 9 and 10
Sample 2: 11 and 12

Sample 1: 13 and 14
Sample 2: 15 and 16

3

Sample 1: 1
Sample 2: 2
Sample 3: 3 and 4

Sample 1: 5
Sample 2: 6
Sample 3: 7 and 8

Sample 1: 9
Sample 2: 10
Sample 3: 11 and 12

Sample 1: 13
Sample 2: 14
Sample 3: 15 and 16

4

Sample 1: 1
Sample 2: 2
Sample 3: 3
Sample 4: 4

Sample 1: 5
Sample 2: 6
Sample 3: 7
Sample 4: 8

Sample 1: 9
Sample 2: 10
Sample 3: 11
Sample 4: 12

Sample 1: 13
Sample 2: 14
Sample 3: 15
Sample 4: 16

5

Sample 1: 1
Sample 2: 2
Sample 3: 3
Sample 4: 4
Sample 5: selectany group from theremaining threegroupsSample 1: 5
Sample 2: 6
Sample 3: 7
Sample 4: 8
Sample 5: selectany group from theremaining threegroupsSample 1: 9
Sample 2: 10
Sample 3: 11
Sample 4: 12
Sample 5: selectany group from theremaining threegroupsSample 1: 13
Sample 2: 14
Sample 3: 15
Sample 4: 16
Sample 5: selectany group from theremaining threegroups66

APPENDIXSTEREO-CITE PROTEO-TRANSCRIPTOMICS SET User ManualSample/lane Method 1

Method 2

Method 3

Method 4

6

Sample 1: 13
Sample 1: 9
Sample 1: 5
Sample 1: 1
Sample 2: 14
Sample 2: 10
Sample 2: 6
Sample 2: 2
Sample 3: 15
Sample 3: 11
Sample 3: 7
Sample 3: 3
Sample 4: 16
Sample 4: 12
Sample 4: 8
Sample 4: 4
Sample 5 and 6:
Sample 5 and 6:
Sample 5 and 6:
Sample 5 and 6:
select any two
select any two groups select any two groups select any two groups
groups from thefrom the remaining from the remaining from the remaining
remaining three
three groups
three groups
three groups
groups7

Sample 1: 1
Sample 2: 2
Sample 3: 3
Sample 4: 4
Sample 5 and 7:
select groups byreferencing themethods used for 3
samples/lane

8

Select any two groups from the four groups.

Sample 1: 5
Sample 2: 6
Sample 3: 7
Sample 4: 8
Sample 5 and 7:
select groups byreferencing themethods used for 3
samples/lane

Sample 1: 9
Sample 2: 10
Sample 3: 11
Sample 4: 12
Sample 5 and 7:
select groups byreferencing themethods used for 3
samples/lane

Sample 1: 13
Sample 2: 14
Sample 3: 15
Sample 4: 16
Sample 5 and 7:
select groups byreferencing themethods used for 3
samples/lane

Perform the following steps:
1. Classify samples 1 to 8 as a group, and add the PCR Barcode Primer Mix by(N, number referencing the methods used for 8 samples/lane.
2. Classify the remaining samples as a group, and correspondingly add differentof mixedgroups of PCR Barcode Primer Mix based on a value of (N-8) by referencing thesamples)methods used for 1 to 8 samples/lane.

N= 9~16

Examples of the mixture with different PCR Barcode Primer Mix:
Example one, 2 samples /lane (refer to Method 1):
1. Take 12.5 μL of PCR Barcode Primer Mix 1 and 2, mix them in equal volumes,
and add them to Sample 1.
2. Add 12.5 μL of PCR Barcode Primer Mix 3 and 4 to Sample 2.
Example two, 13 samples/lane:
1. Add 25 μL of PCR Barcode Primer Mix 1 to Sample 1, 25 μL of PCR Barcode
Primer Mix 2 to Sample 2, ......, and 25 μL of PCR Barcode Primer Mix 12 to Sample
12.
2. Take 6.25 μL of PCR Barcode Primer Mix 13, 14, 15, and 16, respectively, andmix them in equal volumes and add them to Sample 13.

When the library data amount requirements are different:
Libraries that require more than 20% data in a lane are required to use grouped PCR
Barcode Primer Mix
Example:If there are 9 samples pooled in a lane, and 1 of them requires 30% data, thefollowing scheme is required: if the other 8 samples each use PCR Barcode Primer
Mix 1~8, this sample should not use a single PCR Barcode Primer Mix, but use a nonduplicative and grouped PCR Barcode Primer Mix 9~12 or 13~16.
67

APPENDIXSTEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

Appendix C: Antibody Titration
TotalSeq™-A Antibody selection is the key component for combining antibodies of yourinterest in proteo-transcriptome co-detection experiment, and antibody performancecan directly affect data quality. For the Stereo-seq proteo-transcriptome co-detection
experiment, the selection of antibodies follows the selection principle of conventionalmIF method, which requires considerations of host sources, specificities and species
reactivities of added antibodies. We recommend users to first perform antibody
titration for each antibody on tissue-mounted glass slides individually to find the
optimal antibody concentration, then perform a pilot experiment with the selected
antibody concentrations before proceeding to Stereo-seq proteo-transcriptome codetection assay.

Experimental Preparation
Unless otherwise specified, nuclease-free water is used for all reagents being
prepared prior to this experiment.

Reagent/
Consumables

Preparation Steps

Maintenance

5X SSC

Dilute 5 mL of 20x SSC to 20 mL

Room
Temperature

0.1X SSC

Dilute 100 μL of 20x SSC to 20 mL

Room
Temperature

Aliquot Serum

Thaw serum then filter it with a 0.22μm
pore-sized filter and a sterilized syringe.
Aliquot the filtered serum and store at-20°C.

20°C

Microscope Slides

Prepare 6 glass slides for each antibody(5 different concentrations and 1 negative
control).

Room
Temperature

It is recommended to set 2 concentrations below and over the recommended
dilution ratio according to manufacturer's instruction of each antibody. Take theprimary antibody of NeuN (abcam, ab104224) as an example. If the instruction
recommended 1:1000, then set the experimental group with 5 dilution ratios,
1:100, 1:500, 1:1000, 1:5000, 1:1000 and a negative control group (sameprocedures only without the addition of the primary antibody). A total of 6 slides
will be required.

68

APPENDIXSTEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

Reagent

Preparation Steps

Maintenance

4% PFAMix well after thawing and aliquot to
2mL/tube for storage. Equibrate to room
temperature before use.

-20°C for no morethan 1 month

5X SSC

Dilute 5 mL of 20x SSC to 20 mL

Room
Temperature

0.1X SSC

Dilute 100 μL of 20x SSC to 20 mL; Dilute 250 RoomμL of 20x SSC to 50 mL
Temperature

Take the serum out in advance and thaw
it. Mix horse serum and goat serum in a
1:1 ratio. Filter the mixture using a 0.22
μm filter membrane (compatible with asyringe-style sterile disposable syringe).
Recommend aliquoting 200 μL per tube (forFiltered Serum Buffer one round of permeabilization and proteo- -20°C
transcriptomics workflow). Before theexperiment, thaw the aliquoted serum at-20°C. Centrifuge at 4°C, 14000g for 10 min
and keep it for later use. Approximately 20
μL of serum is needed for one chip, and any
remaining serum can be reused.
Do not freeze and thaw the aliquot more than 3 times.
Salmon Sperm DNA

Thaw on ice before use, 20µL/chipPrimary & Secondary
Antibody

Thaw on ice following the original productinstructions, centrifuge for 10 min at
4°C or -20°C
14000g. The Primary Abs should include theisotype Abs

Diluted Primary &Dilute the antibodies if needed.
Secondary (Optional)
10% Triton X-100-20°C

Place on ice until
ready to use

Dilute 100% Triton X-100 into 10% Triton
Room
X-100 with Nuclease-free water if there is noTemperature
ready-to-use 10% Triton X-100

For mouse samples, use TruStain Fcx™ PLUS
FcR Blocking Reagent (CD16/32) Antibody; for human samples,
4°C
use TruStain Fcx™
GlycerolTake it out in advance and equilibrate toroom temperature at least 5 min prior to
use.

Room
Temperature

Decrosslinking
Reagent

Take it out in advance and equilibrate toroom temperature at least 5 min prior to
use.

Room
Temperature

69

APPENDIXSTEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

Other Preparation
Equipment

Set up

Note

Cryostat

Set the cryostat chamber temperature to-20°C and specimen disc temperature(object temperature) to -10°C ~ -15°C.

The specimen
disc temperaturedepends on thetissue type.

PCR Thermal Cycler

Set the temperature to 37°C and hot lid to
42°C.

Check if there is any
abnormality with
the PCR thermal
cycler and replace
it if necessary.

Fluorescence
Microscope

Please select thechannels according
to the fluorescent
Ensure that the microscope is equipped withemission
at least DAPI, FITC, TRITC, CY5
wavelengths ofyour secondary
antibodies.

Centrifuge

Cool the centrifuge temperature to 4 °C in
advance.

-

For guidelines on sample embedding, sectioning, and mounting, please refer toSample Preparation, Sectioning, and Mounting Guide for Fresh Frozen Samples on
Stereo-seq Chip Slides (Document No.: STUM-SP001).
During the temperature equilibration step at cryosection preparation, prepare theblocking buffer by referring to Table C-1 and put it on ice. Meanwhile, take enough
4% PFA solution (at least 400 μL 4% PFA solution per chip) from 4°C to equilibrate
to room temperature.
Table C-1 Blocking Buffer
Components

1X ( µL)

2X + 10% ( µL)

3X + 10% ( µL)

4X + 10% ( µL)

5X SSC

180

252

372

492

10% Triton X-100

120

4.2

6.2

8.2

Blocking Reagent

2

42

62

82

21

31

41

42

62

82

Salmon Sperm DNA,
20
shearedFcR Blocking
10
Reagent*
RI

20

58.8

86.8

114.8

Filtered Serum

28

420

620

820

Nuclease-free water 200

25.2

37.2

49.2

Total

630

930

1230

300
70

APPENDIXSTEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

FcR Blocking Reagent is for blocking of Fc receptors on the cell membrane. Please
select either one according to the host species of your sample. For human tissue,
please use Catalog# 422031 and for mouse tissue, please use Catalog# 156604.
1X blocking buffer is enough for blocking and preparation of Primary antibody
incubation buffer of one tissue section.

Tissue Mounting on Microscopic Slidesa.

Add over 1/3 Volume of 4% PFA to the slide container and ensure that the volume
can cover the tissue on the chip and slide. Prepare enough 4% PFA in a 50 mL
corning tube or an empty slide container at a volume that could submerge the entire
microscopic slide (about 1/3 of the container).

b.

b. Tissue mounting: 1 section per 1 slide is recommended (2 sections per antibody,as 1 section is for the negative control).

c.

c. Tissue mounting can be achieved via either the cold method (option A) or warm
method (option B).
A. Cold Method
1) Place microscope slide inside the chamber with the front-side facing up andpre-cool inside the cryostat chamber for 1~6 min.
Prolonged cooling for over 6 min may cause mist formation on the chip surface.
2) Perform cryosection, then carefully flatten the tissue section out by gently
touching the surrounding OCT with cryostat brushes. Place a tissue section onto
the center of the slide carefully with forceps and brushes. Ensure that the tissue
section is complete and without wrinkles.
3) Immediately pick up the microscope slide and place a finger on the backsideof the microscope slide directly under the tissue for a few seconds to allow thesection to adhere to the surface.
4) Once all tissue mounting is complete, immediately dry the microscope slide at
37°C on a PCR thermal cycler with PCR adaptor for 5 min.
When performing cold mounting, mind the time interval between each tissue
section placement. Longer time intervals (>5 min) could result in tissue wrinkle
formation.
B. Warm Method
1) Perform cryosection and carefully flatten the tissue sections out by gently
touching the surrounding OCT with cryostat brushes.
2) Move the tissue section to the edge, flip the microscope slide and aim thetissue section onto the center of the slide by gently touching the section with themicroscope slide.
3) Check and see if the tissue section has been mounted on to the microscopeslide.
4) Turn the microscope slide over, and immediately dry it at 37°C on a PCR
thermal cycler with PCR adaptor front-side up for 5 min.
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APPENDIXSTEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

Tissue Fixationa.

After drying, immediately immerse the tissue-mounted microscopic slide in 4% PFA
solution at room temperature and fix for 10 min.

b.

After fixation, move the 4% PFA container to a fume hood. Meanwhile, prepare a
50mL centrifuge tube filled with at least 30mL of 0.1X SSC.

c.

Take the microscopic slide out of the 4% PFA solution and immediately immerse theslide in a 50 mL centrifuge tube with at least 30 mL of 0.1X SSC for 10 sec.
Slide transfer should be done as soon as possible to avoid tissue from over drying.

d. Take out the slide and immediately wipe off excess SSC solution from around andthe back of the slide with Kimwipes without touching the tissue to ensure that there
is no residual liquid surrounding the tissue.

e.

Use a Super PAP Pen (hydrophobic barrier pen) on the microscope slide to draw
a circle around the tissue on the slide, creating a hydrophobic isolation zone thatprevents subsequent additional fluid outflow.
Subsequent fluid changes are carried out in a hydrophobic isolation area.
Transfer the processed slides to an immunohistochemistry wet cassette.

f.

All the liquid must be added until the hydrophobic isolation dries.

Tissue Blocking and Primary antibody incubationa.

Vortex the blocking solution that was prepared in Table 3-1 and add no more than
100 μL/section of blocking solution dropwise on the tissue surface within thehydrophobic zone. Incubate for 20 min at room temperature.
The amount of blocking solution used per section is dependent on the size of thehydrophobic area. For a hydrophobic area size of 0.5cm × 0.5cm, the recommended
blocking solution volume is 30 μL/section.

b.

While waiting for the incubation to be done, prepare the Primary Antibody
Incubation Buffer according to Table C-2. Vortex the buffer and leave it on ice until
use.
Take CD68 antibody as an example, antibody titration tests are set for 1:100, 1:250,
1:500 and negative control without primary antibody.
Table C-2 Primary Antibody Incubation Buffer (for Antibody Titration)

Components

1X ( µL)

Primary antibody or diluted primary
antibody*

V^

Blocking Solution

100-V

Total

100
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APPENDIXSTEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual^The amount of primary antibody required is dependent on the dilution ratio.
*If the volume required for the primary antibody is lower than the lowest nominal
capacity of the pipette, the primary antibody should be diluted in advance with theblocking solution.
Discard the blocking solution with a pipette.

c.

•

For experimental groups: Slowly add the primary antibody solution from thenon-tissue area until the solution covers the tissue section. Do not exceed 100
μL/slide. Ensure that to label the dilution ratio on the microscope slide. Incubate
at room temperature for 45 min.

•For negative control group: Add 100 μL/slide of blocking solution. Incubate atroom temperature for 45 min.

Make sure not to dry the tissue during the solution addition process. Otherwise,dried tissue might generate non-specific signals which interferes with the final
results.
d. While waiting for the primary antibody incubation to be done, prepare

secondary antibody solution according to Table C-3. After vortex mixing and brief
centrifugation, leave the secondary antibody solution on ice in the dark until use.
Table C-3 Secondary antibody Incubation Buffer (for Antibody Titration)

Components

1X ( µL)

Secondary antibody

0.2

Blocking Solution

99.8

Total

100

We recommend using Alexa Fluor Plus Series Fluorescent Secondary Antibodies
at a 1:500 dilution concentration. If fluorescent secondary antibodies from othervendors were used, please adjust the dilution ratio according to the manufacturer's
instructions.

Secondary Antibody Incubationa.

Aspirate the primary antibody incubation solution of the experimental group andthe blocking solution of the negative control group.

b.

Add 100 μL 0.1X SSC per section. Incubate at room temperature for 1 min and thenaspirate.

Make sure not to dry the tissue during the solution addition process. Otherwise,dried tissue might generate non-specific signals which interferes with the final
results.

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APPENDIXSTEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

Repeat step b. once.

c.

d. Slowly add no more than 100 μL secondary antibody incubation solution persection from the non-tissue area in both the experimental group and the negative
control group. Incubate for 15 min at room temperature in the dark.

e.

Aspirate the secondary antibody incubation solution.

f.

Add 100 μL 0.1X SSC per section. Incubate at room temperature for 1 min and thenaspirate.

g.

Repeat step f. once.

h.

Hold on to the slide with one hand and completely dry the tissue further with apower dust remover in the other hand at a distance 2-3 cm away from the tissue
surface by blowing gently from one side at a 30-degree angle horizontal to the planeof the slide.

i.

Pipette 5 µL glycerol gently onto the center of the tissue on each chip without
introducing air bubbles.

j.

With a pair of forceps, place one end of the coverslip onto the tissue edge while
holding the other end and then gradually lower the coverslip onto the tissue.
Ensure that the tissue is completely covered by glycerol and the coverslip. To avoid
fluorescent bleaching, IMMEDIATELY proceed to imaging.
Make sure the coverslip is clean without any dust or debris. Wiping with an alcoholswab or blowing with a power dust remover could be used for cleaning.

Imaginga.

Fluorescence images are taken using a fluorescence microscope with image stitching
function, and the fluorescence configuration is recommended as following:•

Light source wavelength range: 380-680 nm•

Monochrome camera (≥ 12 bit)•

DAPI filter cube (ref. Excitation 375/28 nm, Emission 460/50 nm)•

FITC filter cube (Reference: Excitation 480/30 nm, Emission 525/50 nm)•

TRITC filter cube (ref. Excitation 545/25 nm, Emission 605/70 nm)•

CY5 filter cube (Reference: Excitation 620/50 nm, Emission 690/50 nm)•

Maximum pixel size: 5 μm/pixel•

Exposure time: 1 ms~2 s (depending on the characteristics of the antibody used)b.

Transfer the slides to the microscope stage and remove the light shield.

c.

Select the epifluorescence scanning mode, adjust the fluorescence channel
according to the fluorescence excitation light of the secondary antibody used, andscan using 10X lens. After scanning, save both the FOV images and stitched image.
Image all experimental groups with the same imaging parameters to compare thesignal differences across groups.
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APPENDIXSTEREO-CITE PROTEO-TRANSCRIPTOMICS SET User Manual

Guidelines for Selecting Optimal Antibody Concentration
The principle of optimal antibody concentration selection is to select the antibody
concentration that results in the best fluorescent signal of desired cells while minimizing
nonspecific background staining.
Taking the CD68 antibody titration sample data as an example, as shown in the
figure below, select the lowest concentration that does not significantly reduce thefluorescence intensity based on the fluorescence signal. In this example, 1:250 should
be selected as the optimal antibody dilution concentration for subsequent IF pilot
experiments and the official Stereo-CITE Proteo-Transcriptoics experiments.

Ctrl

1:100

1:250

1:500

Figure 10. Immunofluorescent staining of the control and anti-CD68 antibody in mouse thymus

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