Stereo-seq PERMEABILIZATIONSET FOR STEREO-CITE
PROTEO-TRANSCRIPTOMICSAPPLICATIONUSER MANUAL

Cat. No.: 211SP118 (8 RXNs)
Kit Version: V1.0
Manual Version: B

STUM-PR004

Stereo-seq PERMEABILIZATION SET FOR STEREO-CITE PROTEO-TRANSCRIPTOMICS APPLICATION User Manual

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

A
V1.0
Mar. 2024
Initial release

Manual Version:
Kit Version:
Date:
Description：

B
V1.0
May. 2024• Typo fixation• Removal of operational instruction for keeping the chipmoist when removing solution in Section 3.4• Modified Wash Buffer preparation steps in Section 2.1

Stereo-seq PERMEABILIZATION SET FOR STEREO-CITE PROTEO-TRANSCRIPTOMICS APPLICATION User Manual

Note: Please download the latest version of the manual and use it with thecorresponding Stereo-seq Permeabilization 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
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own their intellectual property rights according to law, including but not limited to
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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-seq PERMEABILIZATION SET FOR STEREO-CITE PROTEO-TRANSCRIPTOMICS APPLICATION User Manual

WORKFLOW

Stereo-seq PERMEABILIZATION SET FOR STEREO-CITE PROTEO-TRANSCRIPTOMICS APPLICATION User Manual

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

1
1
3
6
9

CHAPTER 2: SAMPLE AND EXPERIMENTAL PREPARATION
2.1. Experimental Preparation					12CHAPTER 3: FRESH FROZEN SAMPLE, Stereo-seq
PERMEABILIZATION SET FOR STEREO-CITE PROTEOTRANSCRIPTOMICS APPLICATION, STANDARD OPERATINGPROCEDURE
3.1. Tissue Fixation							153.2. Blocking and mock antibody incubation			
16
3.3. Tissue Decrosslinking						
173.4. Tissue Permeabilization Testing				
19
3.5. Reverse Transcription						213.6. Tissue Removal 						223.7. Imaging				24
3.8. Permeabilization Time Determination			25

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-seq PERMEABILIZATION SET FOR STEREO-CITE PROTEO-TRANSCRIPTOMICS APPLICATION User Manual

1.1. Intended Use
STOmics Stereo-seq Permeabilization Set for Stereo-CITE Proteo-Transcriptomics
application enables in situ capture of whole transcriptome and proteomic information
simultaneously and is used for optimizing permeabilization conditions for a specifictissue of interest prior to STOmics Stereo-CITE Proteo-Transcriptomics experiments.
Featured with high resolution and large Field of View (FOV), Stereo-seq Chip P Slides arepatterned with capture probes for capturing mRNA and Antibody Derived Tags (ADT).
Upon interacting with the tissue section, cDNA is synthesized in situ using fluorescently
labeled nucleotides from captured mRNA. Through visualization using fluorescent
microscopy, the optimal permeabilization time can be determined for a specific tissue ofinterest and will be required for further Stereo-CITE Proteo-Transcriptomics Set for Chipon-a-slide experiments.
All reagents provided in this kit have passed stringent quality control and functionalverification, ensuring performance stability and reproducibility.

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

Stereo-seq Permeabilization Kit *1 (8 RXN)•

Stereo-seq Chip P Slide (1cm*1cm) *1 (8 EA)•

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

•
•
•
•
•
•

Additional reagents required:•Decrosslinking Reagents from Stereo-seq Protein Assisted Kit (212KA11)

For more information, refer to Stereo-CITE PROTEO-TRANSCRIPTOMICS SET USERMANUAL
Compatible auxiliary not included:•

Stereo-seq PCR Adaptor (2 EA)

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

Stereo-seq PERMEABILIZATION SET FOR STEREO-CITE PROTEO-TRANSCRIPTOMICS APPLICATION User Manual

Upon receiving the Stereo-seq Chip P Slide (1cm*1cm), follow the instructionsin Stereo-seq Chip Slide Operation Guide For Receiving, Handling And Storing toproperly store unused Stereo-seq Chip P 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 Permeabilization Kit

Stereo-seq Permeabilization Kit

Cat. No.:111KP118

Component

Reagent Cat. No.

Cap Color

RI

1000028499

300 µL × 1

PR Enzyme

1000028500

10 mg

RT QC Reagent

1000028501

748 µL × 1

RT Additive

1000028502(transparent)

44 µL

×1

RT QC Enzyme

1000028503(transparent)

44 µL

×1

TR Enzyme

1000028504

71 µL

×1

TR Buffer

1000028505

1725 µL × 2
Transported
by cold chain

Storage Temperature:-25°C~-18°C

Quantity (tube)

×1

Expiration Date:refer to label

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

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

Cat. No.: 210CP118

Component

Quantity (kit)Stereo-seq Chip P Slide (1cm * 1cm)

8 EA
Transported
by cold chain

Storage Temperature:-25°C~-18°C

2Expiration Date:refer to label

1. INTRODUCTION

Stereo-seq PERMEABILIZATION SET FOR STEREO-CITE PROTEO-TRANSCRIPTOMICS APPLICATION User Manual

Table 1-3 STOmics Accessory Kit

STOmics Accessory Kit

Cat. No.: 1000033700

Component

Reagent Cat. No.

Quantity (per kit)

Cassette

10000033699

1 EA

Gasket10000033698

4 EA

Sealing Tape

1000042970

6 EA

Storage Temperature:
Room Temperature

Transported atroom temperature

Expiration Date:refer to label

Table 1-4 Stereo-seq PCR Adaptor

Stereo-seq PCR Adaptor

Cat. No.: 301AUX001

Component

Quantity (per kit)

Stereo-seq PCR Adaptor

2 EA

Storage Temperature:
Room Temperature

Transported atroom temperature

Expiration Date:refer to label

Table 1-5 Stereo-seq Protein Assisted Kit

Stereo-seq Protein Assisted Kit

Cat. No.:212KA114

Component

Reagent Cat. No.

Decrosslinking
Reagents

1000043548

1725 µL × 5

ADT Amplification Kit 1000043547

220 µL × 1

ADT Primer Mix

1000043548

36 µL

×1

Blocking Reagents

1000044666

60 µL

×1

Storage Temperature:-25°C~-18°C

Cap Color(transparent)
Transported
by cold chain

Quantity (tube)

Expiration Date:refer to label

Reagents in gray are not used in this protocol.

1.3. Additional Equipment and Materials
The tables below (next page) list equipment and materials needed for this protocol.
Some common laboratory equipment not named in Table 1-5 are expected to beaccessible by the user; for instance, an ice maker, biological safety cabinet, freezers, andso on. For specific microscope requirements, refer to STOmics Microscope Assessment
Guideline.
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1. INTRODUCTION

Stereo-seq PERMEABILIZATION SET FOR STEREO-CITE PROTEO-TRANSCRIPTOMICS APPLICATION User Manual

Table 1-6 Additional equipment required

Equipment
Brand

Description

Cat. No.

-

Cryostat--Benchtop centrifuge--

Pipettes-

Metal heating block dry bath (optional) -Vortex mixer-

Eppendorf

Microcentrifuge(for Stereo-seq mIF application,
refrigeration function required)

5418 R

Bio-Rad*T100 Thermal Cycler

1861096

ABI*

ProFlex 3 x 32-well PCR System

4484073

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

Table 1-7 Addition reagents required

Reagents
Brand

Description

Cat. No.

BOSTER (or other brands)

4% Paraformaldehyde (with DEPC)

AR1069

Nuclease-free water

AM9937

1X TE buffer, pH 8.0

AM985820X SSCAM9770Hydrochloric acid, HCl (0.1N)

2104-50ML

Methanol

34860-1L-R

RiboLock RNase Inhibitor (40 U/µL)

EO0382Gibco™ Horse Serum

26050070

Gibco™ Goat Serum

16210064

Invitrogen

Salmon Sperm DNA, sheared(10mg/mL)

AM9680SAKURASAKURA Tissue-Tek® O.C.T. compound 4583

AmbionSigma Aldrich

Thermo Fisher Scientific™

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1. INTRODUCTION

Stereo-seq PERMEABILIZATION SET FOR STEREO-CITE PROTEO-TRANSCRIPTOMICS APPLICATION User Manual

Table 1-8 Additional consumables required

Consumables
Brand

Description

Cat. No.

-

Stainless-steel base mold--

Aluminum foil--

Forceps--

Slide staining rack--

Sterilized Syringe--

Microscope glass coverslip (area: 18 mm
x 18 mm, thickness: 0.13 - 0.16 mm)

Millipore

Millex Syringe Filter, Durapore PVDF,
0.22 µm pore size(for Stereo-seq mIF application)

SLGV033N

Corning® 100 mm TC-treated
Culture Dish

35300350 mL centrifuge tubes

43082915 mL centrifuge tubes

430791BBI

5.0 mL Centrifuge Tubes

F611888-0001

Kimtech

KimWipesTM delicate task wipes

34155

MATIN

Power Dust Remover

M-63181,000 µL filter tips

TF-1000-L-R-S

200 µL filter tips

TF-200-L-R-S

100 µL filter tips

TF-100-R-S

10 µL filter tips

TXLF-10-L-R-S

Invitrogen

Qubit Assay Tubes

Q32856

BIOSHARP

Metal Block-Corning

Axygen5

1. INTRODUCTION

Stereo-seq PERMEABILIZATION SET FOR STEREO-CITE PROTEO-TRANSCRIPTOMICS APPLICATION User Manual

1.4. Practice Tips
Stereo-seq Chip P Slide
Includes 2 Stereo-seq Chip P Slides containing four Chip P (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.

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

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.

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1. INTRODUCTION

Stereo-seq PERMEABILIZATION SET FOR STEREO-CITE PROTEO-TRANSCRIPTOMICS APPLICATION User Manual

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

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.

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

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.

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1. INTRODUCTION

Stereo-seq PERMEABILIZATION SET FOR STEREO-CITE PROTEO-TRANSCRIPTOMICS APPLICATION User Manual

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 yourwhile right thumb between tab 3 and tab 4.

g.

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.

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.

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1. INTRODUCTION

Stereo-seq PERMEABILIZATION SET FOR STEREO-CITE PROTEO-TRANSCRIPTOMICS APPLICATION User Manual

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.

1.5. Precautions and Warnings•

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.
9

1. INTRODUCTION

Stereo-seq PERMEABILIZATION SET FOR STEREO-CITE PROTEO-TRANSCRIPTOMICS APPLICATION User Manual•

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 contaminationof 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 areaand execute regular cleaning (with 0.5% sodium hydrochloride or 10% bleach) toensure 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.

10

1. INTRODUCTION

CHAPTER 2
SAMPLE AND EXPERIMENTPREPARATION

Stereo-seq PERMEABILIZATION SET FOR STEREO-CITE PROTEO-TRANSCRIPTOMICS APPLICATION 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).

2.1. 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 2-20°C for no more
mL/tube for storage. Equibrate to room
than 1 monthtemperature before use.

5X SSC

Dilute 5 mL of 20x SSC to 20 mL.

RoomTemperature

0.1X SSC

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

RoomTemperature

Wash Buffer

Prepare at least 2100 µL per chip (1995 µL Place on ice until
0.1X SSC with 105 µL RI)
ready to use.

Filtered Serum Buffer

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
(for one round of permeabilization and-20°C
proteo-transcriptomics workflow). Before
the experiment, 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 neededfor one chip, and any remaining serum
can be reused.

Do not freeze and thaw the aliquot more than 3 times.
Place on ice until
ready to use.

RI

Thaw on ice before use.

10% Triton X-100

Dilute 100% Triton X-100 into 10% Triton
Room
X-100 with nuclease-free water if there isTemperature
no ready-to-use 10% Triton X-100

12

3. OPERATING PROCEDURE

Stereo-seq PERMEABILIZATION SET FOR STEREO-CITE PROTEO-TRANSCRIPTOMICS APPLICATION User ManualRoomtemperature for
48 hr (Storing
Prepare at least 2 mL of 0.01N HCl per
longer than 48
sample. Configure HCl to 0.01N. Measure
hr will affect theand ensure that the pH = 2.
desired pH. Use
WITHIN 48 hr ofpreparation.)

0.01N HCl

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 powder-20°Cform), and thoroughly mix the reagent by
pipetting.

DO NOT vortex the permeabilization enzyme. Mix by pipetting before use. Aliquot
this 10X stock solution to prevent freeze-thaw cycles.
1X Permeabilization
Reagent Solution

Place on ice until
Make 1X PR solution (150 µL / chip) byready to use. Can
diluting 10X PR stock solution with 0.01N
be on ice for up to
HCl.
6 hr.

Decrosslinking Reagent

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

Other Preparation
Equipments

Set upNote

Set the temperature in the following order:

PCR Thermal Cycler

37°C for slide drying and permeabilization(heating lid at 42°C);

Check the PCR
Thermal Cycler for
abnormalities. If
42°C for reverse transcription (heating lid at necessary, replaceit.
47°C);
55°C for tissue removal (heating lid at 60°C).

Fluorescence
Microscope

Set the epi-fluorescence channel to TRITC
mode.

Room
Temperature

37°C for pre-warming of PR Enzyme;
Metal Bath

70°C for pre-warming of Decrosslinking
Reagent

Adjust thetemperatureaccordinglyCentrifuge

Cool the centrifuge to 4°C in advance.

-

13

3. OPERATING PROCEDURE

CHAPTER 3FRESH FROZEN SAMPLE,
STEREO-seq PERMEABILIZATIONSET FOR STEREO-CITE PROTEOTRANSCRIPTOMICS APPLICATION,
STANDARD OPERATINGPROCEDURE

Stereo-seq PERMEABILIZATION SET FOR STEREO-CITE PROTEO-TRANSCRIPTOMICS APPLICATION User Manual

3.1. Tissue Fixationa.

Refer to 1.4 Practice Tips instructions on assembling the cassette and gasket onto
the Stereo-seq Chip Slide.

b.

Assemble the Stereo-seq Chip Slide onto the cassette, forming a handheld Stereoseq Slide Cassette. Ensure that the 8 tabs are locked in place and that the cassette istightly securing the sides of the cassette.
Do not touch the front of the chip while assembling the Stereo-seq Slide Cassette.

c.

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.

d. After fixation, remove the sealing tape, tilt the Stereo-seq Slide Cassette slightly atan angle of less than 20°, and remove 4% PFA solution from the corner of the wellusing a pipette while keeping the chip and tissue surface moist.

Add and remove solution from the cornere.

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

f.

Tilt the Stereo-seq Slide Cassette slightly at an angle of less than 20°, and remove
the Wash Buffer from a corner of the well using a pipette, keeping the chip andtissue surface moist.
Do not allow the tissue to dry out during the liquid exchange process. Ensure that
all the tissue sections are completely submerged.

g. Repeat steps e. and f. for one more wash..

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3. OPERATING PROCEDURE

Stereo-seq PERMEABILIZATION SET FOR STEREO-CITE PROTEO-TRANSCRIPTOMICS APPLICATION User Manual

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

a.

Add and remove
solution fromthe corner

Table 3-1 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

RI

15

31.5

46.5

61.5

Filtered Serum

30

63

93

123

Nuclease-free water 72

151.2

223.2

295.2

Total

630

930

1230

b.

300

Mock incubation of the primary antibody: remove the blocking buffer solution froma corner of the well using a pipette, keeping the chip and tissue surface moist. Add
150 μL of blocking buffer per well onto the tissue surface immediately. Incubate thechip at room temperature for 45 min.
Do not allow the tissue to dry out during the liquid exchange process. It is
recommended that you operate the procedure one chip at a time.

c.

Mock incubation of the secondary antibody: During the mock primary antibody
incubation step, prepare the mock secondary antibody solution according to Table
3-2. Vortex the mixture and place it on ice for later use.

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3. OPERATING PROCEDURE

Stereo-seq PERMEABILIZATION SET FOR STEREO-CITE PROTEO-TRANSCRIPTOMICS APPLICATION User Manual

Table 3-2 Secondary Antibody Mock Incubation Buffer

Components

1X ( µL)

2X + 10% ( µL)

3X + 10% ( µL)

4X + 10% ( µL)

5X SSC

90

189

279

369

RI

7.5

15.75

23.25

30.75

Nuclease-free water 52.5

110.25

162.75215.25

Total

315

465

615

150

d. Remove the blocking buffer from a corner of the well using a pipette, and keeping

the chip and tissue surface moist.

e.

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

f.

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, keeping the chip and tissue
surface moist.

g.

Repeat steps e. and f. for one more wash..

h.

Immediately add 150 μL secondary antibody mock incubation buffer per well andincubate the chip in the dark at room temperature for 15 min.

i.

Prepare the decrosslinking reagents during the secondary antibody incubation step.

3.3. Tissue Decrosslinkinga.

Thaw the Decrosslinking Reagent in advance according to the 2.1 Experiment
Preparation section and bring it to room temperature.

b.

Set one module of the PCR Thermal Cycler to 70°C and the lid temperature to 75°C.
Place the PCR Adaptor in the thermal cycler to equilibrate in advance.

c.

Incubate the Decrosslinking Reagent at 70°C for at least 10 min.

d. Remove the mock secondary antibody incubation solution from the corner of thewell while keeping the tissue surface moist.

e.

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

f.

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, keeping the chip and tissue
surface moist.

g.

Repeat steps e.-f. for one more wash.

h.

After the final wash, add 400 μL Decrosslinking Reagent per well.
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3. OPERATING PROCEDURE

Stereo-seq PERMEABILIZATION SET FOR STEREO-CITE PROTEO-TRANSCRIPTOMICS APPLICATION User Manuali.

Seal with sealing tape and then place the Stereo-seq Slide Cassette on the PCR
Adaptor (70°C). Close the lid of the PCR Thermal Cycler and perform decrosslinking
at 70°C for 15 min.

Sealing tape

PCR Adaptorj.

During the decrosslinking process, set aside the 2 mL of 0.01N HCl and 1X
Permeabilization Reagent Solution you prepared in 2.1 Experimental Preparation.

k.

Ensure that the 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.

l.

Temperature

Time

Cycle

(Heated lid) 42°C

on

-

37°C

60 min

1

37°C

Hold

-

Warm the aliquoted 1X Permeabilization Reagent Solution inside the 37°C PCR
Thermal Cycler or on a Metal Block for >10 min (no longer than 30 min).

m. After decrosslinking, transfer the Stereo-seq Slide Cassette to the lab bench, remove

the sealing tape, and cool to room temperature for 10 min.

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Stereo-seq PERMEABILIZATION SET FOR STEREO-CITE PROTEO-TRANSCRIPTOMICS APPLICATION User Manual

3.4. Tissue Permeabilization Testing
For positive control, total RNA or mouse brain tissue (incubate at 37°C for 12 min)can be used.
a.

After cooling, slightly tilt the Stereo-seq Slide Cassette at an angle of less than 20°.
Discard the decrosslinking reagent from the corner of the well using a pipette,
keeping the chip and tissue surface moist. Add 400 μL of Wash Buffer per well.

b.

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.

c.

Tissue sections on the Stereo-seq Chip P Slide are incubated for different lengthsof time in the range of 0-30 min. For the first trial, it is recommended that you use asuggested time course of 8 min, 14 min, 20 min, and 26 min along with a positive
control group (mouse thymus tissue or total RNA).

8 min
14 min20 min26 minFigure 1. Permeabilization Times (min)Keep the Stereo-seq Slide Cassette in the PCR Thermal Cycler during thepermeabilization and reverse transcription steps. Do not move the cassette.

Addsolutionto themiddle1) Place the Stereo-seq Slide Cassette in the 37°C PCR Thermal Cycler. Add 150 μL
of 1X Permeabilization Reagent Solution onto the chip (with 26-min time point) by
first pipetting one droplet at each corner of the chip and then adding the rest of thesolution to the middle to merge all droplets.
Ensure that the chip is completely covered with 1X Permeabilization Reagent
Solution.
2) Apply unpeeled sealing tape to the Stereo-seq Slide Cassette and let the chipincubate inside the PCR thermal cycler at 37°C.
3) After 6 min, open the lid, remove the unpeeled sealing tape and add 150 μL of 1X
Permeabilization Reagent Solution on the chip (with 20-min time point).
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4) Apply unpeeled sealing tape to the Stereo-seq Slide Cassette, close the lid andincubate at 37°C.
5) Repeat the process, working backwards to the shortest incubation time (chip with
8-min time point).
A second trial of permeabilization time determination (3 more time points) might
be required depending on the first trial’s results. If multiple time points show
similar fluorescent signals, we suggest choosing longer time points for your second
trial.
d. For total RNA as positive control:1) Prepare the Total RNA Hybridization Mix as indicated in Table 3-3.
Table 3-3 Total RNA hybridization Mix
Components

1X ( µL)

Total RNAX (2µg)Nuclease-free water

70-X

20X SSC

25

RI

5

Total

100

2) Warm up the Total RNA hybridization Mix inside the 37°C PCR thermal cycler for>3 min.
3) Add 100 µL of total RNA hybridization mixture onto a chip and incubate at 37°C for
15-20 min.
Tissue removal is not needed for total RNA as the positive control group.
e.

When incubation is completed, remove the Stereo-seq Slide Cassette from the PCR
Adaptor.

f.

Slightly tilt the Stereo-seq Slide Cassette, remove 1X Permeabilization Reagent
Solution or total RNA hybridization mixture with a pipette from the corner of eachwell without touching the chip surface.

g.

Add 100 µL of Wash Buffer and then remove the solution from the corner of each
well.

h.

Place the PCR Adaptor in another PCR Thermal Cycler in advance and set thetemperature to 42°C with heated lid set to 47°C.

i.

Prepare RT QC Mix according to Table 3-4 and equilibrate to room temperature in
the dark.

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Table 3-4 RT QC Mix
Components

1X ( µL)

2X + 10% ( µL)

3X + 10% ( µL)

4X + 10% ( µL)

RT QC Reagent

85

187

280.5

374

RT Additive

5

11

16.5

22

RT QC Enzyme

5

11

16.5

22

RI

5

11

16.5

22

Total

100

220

330

440

3.5. Reverse Transcription
a. Ensure that the temperature of the PCR Thermal Cycler with PCR Adaptor has been set
to 42°C in advance.
b. Gently add 100 µL of RT QC Mix per chip along the side of each well, ensuring that thewell surface is uniformly covered with RT QC Mix.
c. Apply sealing tape to the Stereo-seq Slide Cassette and ensure that it is sealed tightly.
Incubate the Stereo-seq Slide Cassette at 42°C for 1 hr or longer (no longer than 16 hr) in
the dark with the following incubation protocol.

Temperature

Time

Cycle

(Heated lid) 47°C

on

-

42°C

60 min

1

42°C

Hold

-

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3.6. Tissue Removal
PrepareReagent

Preparation Steps

StorageTR buffer

Heat the buffer for 5 min at 55 °C todissolve the precipitate. Equilibrate it
to room temperature prior to use.

Room Temperature

If white precipitate is observed in the buffer, dissolve it by heating the buffer at 55°C
again and equilibrate to room temperature before mixing.
a.

Ensure that the PCR Thermal Cycler with PCR Adaptor has been set to 55°C and theheated 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 the Stereo-seq Slide Cassette with one hand
without applying force to Side A and Side B of the cassette. Doing so prevents the
Stereo-seq Chip Slide from falling off of the cassette.

c.

Slightly tilt the Stereo-seq Cassette and remove the RT QC Mix with a pipette fromthe corner of each well without touching the chip surface.

d. Add 400 µL of 0.1X SSC solution into each well.
e.

Gently pipette 0.1X SSC solution up and down at the corner of each well 5 times.

f.

Slightly tilt the Stereo-seq Cassette and remove 0.1X SSC with a pipette from thecorner of each well.

g.

Repeat step d.-f.

h.

Prepare the Tissue Removal Mix as shown in Table 3-5.

Table 3-5 Tissue Removal Mix
Components

1X ( µL)

2X + 10% ( µL)

3X + 10% ( µL)

4X + 10% ( µL)

TR Buffer

392

862.4

1293.6

1724.8

TR Enzyme

8

17.6

26.4

35.2

Total

400

880

1320

1760

i. Add 400 µL of Tissue Removal Mix per well without introducing bubbles. Ensure that
there is uniform solution coverage within each well.

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j. Apply sealing tape to the Stereo-seq Slide Cassette and incubate at 55 °C on the PCR
Adaptor for 1 hr with the following incubation protocol.

Temperature

Time

Cycle

(Heated lid) 60°C

on

-

55°C

60 min

1

55°C

Hold

-

k. At the end of incubation, remove the Stereo-seq Slide Cassette from the PCR Adaptor
and remove the sealing tape.
If tissue remains on the chip after the tissue removal step, increase the incubation
time (no longer than 16 hr). Ensure that the tissue is completely removed.
l. Slightly tilt the Stereo-seq Cassette, remove Tissue Removal Mix with a pipette fromthe corner of each well.
m. Add 400 µL of 0.1X SSC solution into each well.
n. Gently pipette 0.1X SSC solution up and down at the corner of each well for 5 times.
o. Remove 0.1X SSC with a pipette from the corner of each well.
p. Repeat step m.-o.
q. Add 400 µL of nuclease-free water into each well and pipette up and down to washthe chip surface as SSC solution contains salt.
r. Remove the slide from the Stereo-seq Slide Cassette according to the instructions in
1.4 Practice Tips.
Alternative Step:
Remove the slide from the Stereo-seq Slide Cassette after step l. and rinse theStereo-seq Chip Slide up and down 10 times in a 50 mL falcon tube filled with 50 mL
0.1X SSC, then rinse up and down 10 times with 50 mL nuclease-free water.
s. Place the Stereo-seq Chip Slide onto a clean dust-free paper and dry the chip surface
completely with a power dust remover (MATIN, M-6318).

If visible tissue traces remain on the surface of the chip, wash again by adding 100µL nuclease-free water and then blow dry. This step can be repeated until no visible
traces remain on the chip surface.
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Stereo-seq PERMEABILIZATION SET FOR STEREO-CITE PROTEO-TRANSCRIPTOMICS APPLICATION User Manualt. Place the Stereo-seq Chip Slide in a clean petri dish and wrap it with aluminum foil.
The chips are now ready for imaging.

3.7. Imaging
a. Create a new folder in a fluorescent microscope-connected PC, name it with the chip
ID number and other essential information.
Only use letters, numbers, and underscores in folder naming. Special charactersand spacings are not allowed.
Example: B00249A1
b. Take fluorescence images from the chip with the following microscope setting: TRITC
channel, 4X and 10X objective lenses, with stitching function.
c. Place 1-2 µL of water on the imaging platform first, then transfer and place the Stereoseq Chip Slide onto the water drop. Water surface tension will grab onto the Slide andcause it to adhere to the imaging platform.

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Stereo-seq PERMEABILIZATION SET FOR STEREO-CITE PROTEO-TRANSCRIPTOMICS APPLICATION User Manuald. Remove the light shield and select the chip area of interest.

e. Find the desired capturing area with the 4X lens first and then switch to 10X lens tocomplete the full scan.
Ensure that the desired capturing area is clear and within focus during fullscanning.
Positive control with RNA extract should be imaged separately without modifying
any parameters.

3.8. Permeabilization Time Determination
The optimal permeabilization time should result in the strongest fluorescence signal
with the lowest signal diffusion. However, this is based on a complete tissue removal aswell as images taken under the same settings. For example, as shown in Figure 2, for the
8 min permeabilization time point, the fluorescence signal in some parts of the mouse
thymus is very low, suggesting insufficient permeabilization. For the 14 min and 20 min
permeabilization time point, images show the strongest signal and finer details amongthe 4 time points. For the 26 min permeabilization time point, the signal is the lowest.
Based on this result, the optimal permeabilization time for this tissue is 14 min and 20
min. In such case when two permeabilization time points show similar fluorescencesignals, we suggest using the shorter time, i.e., 14 min as the optimal permeabilization
time point to avoid potential diffusion.

Figure 2. The optimal permeabilization time determination of a mouse thymus section25

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