Stereo-seq TRANSCRIPTOMICSSET FOR FFPEUSER MANUAL

Cat. No.: 211SN114 (4 RXN)
Kit Version: V1.0
Manual Version: A

STUM-TT004

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manual

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

A
V1.0
Jul. 2024
Initial releaseStereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manual

Note: Please download the latest version of the manual and use it with thecorresponding Stereo-seq Transcriptomics N 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 of 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. does not grant or imply the right or license to use any
copyrighted content or trademark (registered or unregistered) of ours or any third party.
Without our written consent, no one shall use, modify, copy, publicly disseminate,change, distribute, or publish the program or Content of this manual withoutauthorization, and shall not use the design or use the design skills to use or takepossession of the trademarks, the logo or other proprietary information (including
images, text, web design or form) of ours or our affiliates.
4. Nothing contained herein is intended to or shall be construed as any warranty,
expression or implication of the performance of any products listed or described herein.
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-seq TRANSCRIPTOMICS SET FOR FFPE User Manual

WORKFLOW

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE 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. Stereo-seq Chip Slide Information			1.6. Precautions and Warnings 			

1
1
1
4
5
6

CHAPTER 2: SAMPLE AND EXPERIMENT PREPARATIONCHAPTER 3: Stereo-seq TRANSCRIPTOMICS SET FOR FFPE
STANDARD OPERATING PROCEDURE
3.1. Experimental Preparation					103.2. Deparaffinization						133.3. H&E Staining, Imaging,
Decolorization and Decrosslinking				143.4. Fluorescent Staining, Imaging and Decrosslinking
20
3.5. Fixation								243.6. Tissue Permeabilization					253.7. FFPE MIX Reaction 			26
3.8. cDNA Release and Collection				27
3.9. cDNA Purification and Amplification			28
Appendix I: Stereo-seq Slide Cassette Assembly35

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 TRANSCRIPTOMICS SET FOR FFPE User Manual

1.1. Intended Use
STOmics Stereo-seq Transcriptomics Set for FFPE is intended for generating a spatiallyresolved total RNA library from formalin-fixed and paraffin-embedded (FFPE) biologicaltissue sections. Built upon DNA Nanoball (DNB) technology, STOmics Stereo-seq
Transcriptomics Set for FFPE enables a “tissue-to-data” solution through in situ captureof the whole transcriptome, at nanoscale resolution and centimeter-sized field of view(FOV). This kit uses random primers to capture and sequence RNAs in situ . Each cDNA
synthesized from mRNA captured on a particular spot is linked to its spatially-barcoded
probe, allowing subsequent gene expression mapping of a tissue section following
sequencing and visualization analysis using the StereoMap 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 using the Stereo-seq Transcriptomics Set require the
DNBSEQ sequencing platform. For details, refer to Stereo-seq OMNI FFPE Library
Preparation User Manual (Document No.: STUM-LP001)
Required input parameters for Stereo-seq FFPE transcriptome libraries for the Stereoseq Analysis Workflow (SAW) bioinformatics pipelines are as follows:--kit-version= 'Stereo-seq N FFPE V1.0'--sequencing-type = 'PE75_25+59'

1.3. List of Kit Components
Each Stereo-seq Transcriptomics Set for FFPE consists of：•

Stereo-seq Transcriptomics N Kit V1.0 *1 (4 RXN)•

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

STOmics FFPE Accessory Kit *3

Stereo-seq 16 RXN Library Preparation Kit is not included in the Stereo-seq
Transcriptomics Set for FFPE and must be purchased separately. If you wish to construct
Stereo-seq FFPE transcriptome libraries in-house, refer to STUM-LP001 Stereo-seq
OMNI FFPE Library Preparation User Manual for details.

1

1. INTRODUCTION

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manual•

Compatible auxiliary but not included:
Stereo-seq PCR Adaptor *1 (2 EA)

Catalog numbers, kit components, and specifications are listed below (Table 1-1 toTable 1-4).
Upon receiving the Stereo-seq Chip N Slide (1cm*1cm), follow the instructionsin Stereo-seq Chip Slide Operation Guide For Receiving, Handling And Storing toproperly store unused Stereo-seq Chip N 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.

2

1. INTRODUCTION

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manual

Table 1-1 Stereo-seq Transcriptomics N Kit

Stereo-seq Transciptomics N Kit

Cat. No.:211KN114

Component

Reagent Cat. No.

RI

1000028499

300 μL × 1

FFPE Mounting Medium

1000047466

100 μL × 1

FFPE Decrosslinking Reagent 1000047464

1725 μL × 2

PR Enzyme

1000028500

10 mg × 1

FFPE RT Buffer Mix

1000047460(transparent) 700 μL × 1

FFPE RT Oligo

1000047461(transparent) 44 μL × 1

FFPE RT Enzyme Mix

1000047462(transparent) 132 μL × 1

FFPE Dimer

1000047463

10 μL × 1

cDNA Release Buffer

1000028512

1725 μL × 2

cDNA Release Enzyme

1000028511

88 μL × 1

cDNA Amplification Mix

1000028514

220 μL × 1

FFPE cDNA Primer Mix

1000047465

36 μL × 1

Storage Temperature:-25°C~-18°C

Cap Color

Transported
by cold chain

Quantity (tube)

Expiration Date:refer to label

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

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

Cat. No.: 210CN114

Component

Quantity (per kit)

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

4 EA
Transported
by cold chain

Storage Temperature:
2°C~8°C

3

Expiration Date:refer to label

1. INTRODUCTION

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manual

Table 1-3 STOmics FFPE Accessory Kit Components

STOmics FFPE Accessory Kit

Cat. No.: 310AK002

Component

Reagent Cat. No.

Cap Color

Quantity (per kit)

Cassette

1000033699

-

3 EA

Gasket1000033698

-

3 EA

Sealing Tape

1000042970-/

Storage Temperature:
Room Temperature

Transported atroom temperature

Expiration Date:refer to label

Table 1-4 Stereo-seq PCR Adaptor Components

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

1.4. Additional Equipment and Materials
For equipment and materials needed for this protocol, refer to the Experiment
Preparation Checklist for FFPE. The user is expected to have access to common
laboratory equipment not named in the document (equipment such as an ice maker,
biological safety cabinet, freezers, and so on). For specific microscope requirements,
refer to STOmics Microscope Assessment Guideline.

https://en.stomics.tech/resources/documents

4

1. INTRODUCTION

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manual

1.5. Stereo-seq Chip Slide Information
Stereo-seq Chip N Slide
Includes 4 Stereo-seq Chip N Slides containing one Chip N (1cm*1cm) on each slide.
Stereo-seq Chip P Slides, Stereo-seq Chip T Slides, and Stereo-seq Chip N Slides aredifferentiated by a laser-engraved label at the end of the slide.

Stereo-seq Chip N Slide Storage
Always store unused slides in their original slide container and keep them sealed in asealable 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.

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

Refer to Appendix I: Stereo-seq Slide Cassette Assembly5

1. INTRODUCTION

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE 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, ensure that you are familiar with all
related instruments and operate them according to the manufacturers' instructions.

•

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

•Thaw reagents in the kits properly prior to use. For enzymes, centrifuge briefly andkeep them on ice until use. For other reagents, thaw them first at room temperature,
invert several times to mix them properly, and centrifuge them briefly. Place themon ice for future use.

•

RNA capture will be compromised or absent for any scratched areas on the frontside surface of the chip.

•

We recommend using filtered pipette tips to prevent cross-contamination. 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, for PCR
reaction preparation and PCR product cleanup tests, we recommend working in
two distinctly separated working areas in the laboratory. Use designated pipettes
and equipment for each area, and perform 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.

6

1. INTRODUCTION

CHAPTER 2
SAMPLE AND EXPERIMENTPREPARATION

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User ManualFor paraffin sample sectioning and mounting guides, refer to Sample Preparation,
Sectioning, and Mounting Guide for Formalin-fixed and Paraffin-embedded (FFPE)
Samples on Stereo-seq Chip Slides (Document No.: STUM-SP003).
This guide explained how to check the RNA quality (DV200 value) of a FFPE tissue sample
before proceeding to the Stereo-seq experiment.
It is strongly recommended that you proceed only with tissue samples with a
DV200 ≥ 30%.

8

2. SAMPLE AND EXPERIMENT PREPARATIONCHAPTER 3
Stereo-seq
TRANSCRIPTOMICS SETFOR FFPE STANDARD
OPERATING PROCEDURE

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manual

3.1. Experiment Preparation
Unless otherwise specified, use nuclease-free water to prepare all reagents being
prepared prior to this experiment.
Table 3-1 Reagent Preparation Master Planning Table

Prep
Day

Reagent

Workflow
Section

Day 1

400 mL 30%
ethanolTissueDilute anhydrous ethanol tosectioning and
30% using ddH2O.
mounting

Room
Temperature up
to 1 day

100 mL 96%
ethanol

Deparaffinization

Dilute anhydrous ethanol to
96% using ddH2O.

Room
Temperature up
to 1 day

50 mL 90%
ethanol

Deparaffinization

Dilute anhydrous ethanol to
90% using ddH2O.

Room
Temperature up
to 1 day

50 mL 80%
ethanol

Deparaffinization

Dilute anhydrous ethanol to
80% using ddH2O.

Room
Temperature up
to 1 day

50 mL 70%
ethanol

Deparaffinization

Dilute anhydrous ethanol to
70% using ddH2O.

Room
Temperature up
to 1 day

50 mL 50%
ethanol

Deparaffinization

Dilute anhydrous ethanol to
50% using ddH2O.

50 mL 30%
ethanol

Deparaffinization

Dilute anhydrous ethanol to
30% using ddH2O.

Day 2

Preparation Steps

Maintenance

Room
Temperature up
to 1 day
Room
Temperature up
to 1 dayHematoxylin(Solarbio,
H&E staining
G4470)Take it out of 4°C in advanceRoomand equilibrate to room
Temperature up
temperature at least 5 min
to 2 hrprior to use.

Eosin
Solution

Eosin was diluted with 70% Roomethanol at a 2:1 volume ratio. Temperature

H&E staining

50 mL 5X SSC H&E staining
200 mL 0.1X
SSC

H&E staining

10

Room
Add 12.5 mL 20X SSC into 37.5
Temperature up
mL ddH2O and mix well.
to 1 week
Room
Add 1 mL 20X SSC into 199 mL
Temperature up
ddH2O and mix well.
to 1 week

3. FFPE STANDARD OPERATING PROCEDURE

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User ManualRoom
Add 50 μL 20X SSC into 150 μL
Temperature up
200 μL 5X SSC ssDNA staining
ddH2O and mix well.
to 1 week

Day 2

50 mL 0.1X
SSC

ssDNA stainingRoom
Add 250 μL 20X SSC into 49.75
Temperature up
mL ddH2O and mix well.
to 1 week

FFPE
Mounting
Medium

H&e staining
and ssDNA
staining

Take it out of -20°C in advanceRoomand equilibrate to room
Temperaturetemperature until it thaws.

Fluorescent
Staining
SolutionRoom
Add 1 μL Qubit ssDNA
Temperature in
ssDNA staining Reagent and 10 μL RI into 189
the dark up to 1
μL 5X SSC and mix well.
hr

TE buffer(pH=9.0)

Take it out of 4°C in advanceRoomand equilibrate to room
H&E
Temperature up
decolorization temperature at least 5 min
to 2 hrprior to use.

FFPE
Decrosslinking
Reagent

Take it out of -20°C in
advance, equilibrate to room
Room
temperature until it thaws,
Decrosslinking
then mix well to ensure that Temperature
no white precipitates arevisible.

If white precipitates are visible in the reagent, dissolve them by heating
the buffer at > 50 °C and equilibrate to room temperature before mixing.

0.01N HCl

Permeabilization

Room
temperature for
Prepare at least 1 mL of 0.01N 48 hr (Storing
HCl per sample. Configure
longer than 48
HCl to 0.01N. Measure andhr will affectmake sure the pH = 2.
the desired
pH. Please use
WITHIN 48 hr ofpreparation)

ALWAYS use freshly prepared 0.01N HCl (pH = 2.0 ± 0.1). For pre-made 0.1N
HCl and newly purchased HCl, check the pH prior to the experiments.
10X
Permeabilization
Reagent
Stock
Solution

Permeabilization

Add 1 mL of freshly prepared
0.01N HCl to dissolve PR-20°C up to 1
Enzyme (red cap, in powder
monthform), and thoroughly mix thereagent through pipetting.

DO NOT vortex the permeabilization enzyme. Mix by pipette before using.
Aliquot this 10X stock solution to avoid freeze-thaw cycles.

11

3. FFPE STANDARD OPERATING PROCEDURE

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manual

1X
Permeabilization
Reagent
Solution

Permeabilization

Make 1X PR solution (200 μL /On ice until use,chip) by diluting 10X PR stock
up to 6 hrsolution with 0.01N HCl.

0.1X SSC

Permeabilization

Add 10 μL 20X SSC into 1990
μL nuclease-free water andmix well.

0.1X SSC(with 5% RI)

Permeabilization

Add 10 μL RI into 190 μL 0.1X
On ice until use
SSC and mix well

FFPE RT
Buffer Mix

Take it out of -20°C in
advance, equilibrate to room
temperature until it thaws,
RT and ligation
On ice until use
then mix well to ensure thatno white precipitates arevisible.

FFPE Dimer

RT and ligation

Take it out of -20°C in
advance, and thaw on ice.

On ice until use

FFPE RT Oligo RT and ligation

Take it out of -20°C in
advance, and thaw on ice.

On ice until use

cDNA ReleasecDNA Release
BufferTake it out in advance andheat the buffer for 5 min
at 55°C to dissolve theprecipitate. Equilibrate it toroom temperature prior to
use.

Room
temperature

Day 2

Room
Temperature up
to 1 week

If white precipitates are visible in the buffer, dissolve them by heating thebuffer at 55 °C again and equilibrate to room temperature before mixing.

Day 3

Magnetic
beads

cDNA
Purification

Take it out in advanceand equilibrate to room
temperature at least 30 min
prior to use.

10 mL 80%
Ethanol

cDNA
Purification

Room
Dilute 100% ethanol to 80%. temperature up
to 1 day

cDNA
cDNA
Amplification
Amplification
Mix
FFPE cDNA
Primer Mix

cDNA
Amplification

TE buffer, pH cDNA
8.0
Purification

12

4°C

Take it out of -20°C in
advance, and thaw on ice.

On ice until use

Take it out of -20°C in
advance, and thaw on ice.

On ice until use

Set it aside at room
temperature until use

Roomtemperature

3. FFPE STANDARD OPERATING PROCEDURE

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manual

Other Preparation
Equipment

Set upNotes

Set the temperature in the following order:
1. 85°C for H&E decolorization (heated lid at85°C).
2. 95°C for decrosslinking (heated lid at 85°C). Check the PCR
thermal cycler for
PCR Thermal Cycler 3. 37°C for slide baking, permeabilization,
any abnormalities,
and RT ligation (heated lid at 42°C).
and replace it ifnecessary.
4. 42°C for slide baking and RT + ligation(heated lid at 42°C).
5. 55°C for cDNA release (heated lid at 60°C).
For H&E imaging:Set to BF-Epi channel.

Fluorescence
Microscope

For fluorescent imaging:Set the epi-fluorescence channel to FITC
mode

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

3.2. Deparaffinizationa.

Set aside the ethanol of different concentrations and ddH2O you prepared in 3.1
Experiment Preparation, turn on the Slide Dryer in advance (or Water bath-Slide
Dryer, metal bath, or PCR thermal cycler with PCR Adaptor), and set the baking
temperature to 60°C.

b.

Set aside the hematoxylin solution from a 4°C refrigerator and equilibrate it to room
temperature in advance.

c.

Transfer the tissue-mounted Stereo-seq Chip Slide onto the Slide Dryer and bake at
60°C for 1 hr.

d. Prepare the following reagents in staining jars, at least 30 mL for each jar:

Quantity

Description
Histo-clear

2

1 & 2

100% ethanol 1 & 2

2

90% ethanol

2

1 & 2

80% ethanol

1

70% ethanol

113

3. FFPE STANDARD OPERATING PROCEDURE

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manual

Description

Quantity

50% ethanol

130% ethanol

1

ddH2O

1

Xylene can be used in place of Histo-cleare.

After baking, place the Stereo-seq Chip Slide into Histo-clear 1 at room temperaturefor 20 min, then transfer it to Histo-clear 2 at room temperature for another 20 min.

f.

Take the Stereo-seq Chip Slide out from Histo-clear 2 and remove the excess Histoclear solution with dust-free paper.

g.

Sequentially place the Stereo-seq Chip Slide into the following containers:
1. 100% ethanol 1 for 5 min
2. 100% ethanol 2 for 5 min
3. 96% ethanol 1 for 5 min
4. 96% ethanol 2 for 5 min
5. 90% ethanol for 2 min
6. 80% ethanol for 2 min
7. 70% ethanol for 2 min
8. 50% ethanol for 2 min
9. 30% ethanol for 2 min
10.ddH2O for 1 min.

h.

Take the Stereo-seq Chip Slide out and remove excess ddH2O from around and theback of the slide with dust-free paper without touching the chip.

3.3. H&E Staining, Imaging, Decolorization and Decrosslinking
Stereo-seq Transcriptomics for FFPE workflow offers two staining options: H&E
staining or nuclei staining using ssDNA. Please choose one of the staining methods
and proceed to section 3.3 for the H&E staining workflow or section 3.4 for the
fluorescent staining workflow.

14

3. FFPE STANDARD OPERATING PROCEDURE

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manual

Reagent Required
for Section 3.3

Purpose

Preparation

Hematoxylin

Staining

Prepare 1 mL per chip. Take it out of
4°C in advance and equilibrate to room
temperature at least 5 min prior to use.

5X SSC

Washing

Dilute 12.5 mL 20X SSC with 37.5 mL
ddH2O in a staining jar (alternatively,
use a slide container or a 50 mL
centrifuge tube) and mix well.

Bluing reagent

Staining

Prepare 30~40 mL bluing reagent in astaining jar (alternatively, use a slide
container or a 50 mL centrifuge tube).

0.1X SSC

Washing

Prepare 30~40 mL 0.1X SSC in two
staining jars separately (alternatively,
use a slide container or a 50 mL
centrifuge tube).

70% ethanol

Eosin dilution

Dilute 14 mL 100% ethanol with 6 mL
ddH2O and mix well.

Eosin Solution

Staining

Dilute 10 mL Eosin with 20mL 70%
ethanol (Eosin solution can be re-used)
in a staining jar (alternatively, use aslide container or a 50 mL centrifugetube) and mix well.

FFPE Mounting Medium

Take it out of -20°C in advance andChip Mounting equilibrate to room temperature until
it thaws.

0.1X SSC

Washing andcoverslip
detachment

TE Buffer (pH9.0)

FFPE Decrosslinking Reagent

Prepare 30~40 mL 0.1X SSC in astaining jar (alternatively, use a slide
container or a 50 mL centrifuge tube).

Take it out of 4°C in advance andH&E
decolorization equilibrate to room temperature atleast 5 min prior to use.
Take it out of -20°C in advance,
Decrosslinking equilibrate to room temperature until
it thaws, then mix well to ensure thatno white precipitates are visible.

If white precipitates are visible in the reagent, dissolve them by heating the bufferat 55 °C and equilibrate to room temperature before mixing.
15

3. FFPE STANDARD OPERATING PROCEDURE

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manual

H&E Staininga.

To achieve optimal staining results, make sure the hematoxylin solution has beenequilibrated to room temperature.

b.

Pipette enough nuclease-free water on the Stereo-seq Chip then discard it tomoisten the chip surface and facilitate H&E staining. Add 1 mL of hematoxylin
solution onto the Stereo-seq Chip, cover the chip surface completely, and stain for 2
min at room temperature.

c.

After the incubation, discard the hematoxylin solution by turning the Stereo-seq
Chip Slide sideways while pouring out the solution onto dust-free paper.

d. Immerse the Stereo-seq Chip Slide in a staining jar (alternatively, use a slide

container or a 50 mL centrifuge tube) with about 30 mL of 5X SSC, and then gently
rinse the Stereo-seq Chip Slide up and down 5 times to wash away the hematoxylin
solution.

e.

Immerse the Stereo-seq Chip Slide in a staining jar (alternatively, use a slide
container or a 50 mL centrifuge tube) with about 30 mL of the bluing reagent for 1
min at room temperature.

f.

Immerse the Stereo-seq Chip Slide in a staining jar (alternatively, use a slide
container or a 50 mL centrifuge tube) with about 30 mL of 0.1X SSC, and then gently
rinse the Stereo-seq Chip Slide up and down 5 times to wash away the bluing
reagent.

g.

Immerse the Stereo-seq Chip Slide in a staining jar (alternatively, use a slide
container or a 50 mL centrifuge tube) with about 30 mL of Eosin solution for 1 min atroom temperature (the Eosin solution can be reused).

h.

Immerse the Stereo-seq Chip Slide in a staining jar (alternatively, use a slide
container or a 50 mL centrifuge tube) with about 30 mL of 0.1X SSC, and thengently rinse the Stereo-seq Chip Slide up and down 3 times to wash away the Eosin
solution.

16

3. FFPE STANDARD OPERATING PROCEDURE

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manuali.

Securely hold the slide and completely dry the tissue using a power dust remover at
a distance of 2-3 cm away from the tissue surface. Below gently from one side at a30-degree angle horizontal to the plane of the slide.

j.

Pipette 5 µL FFPE Mounting Medium onto the center of the tissue on the chip
without introducing air bubbles.
Make sure the coverslip is clean and free of dust and debris. To clean the coverslip,
wipe it with an alcohol swab or use a power dust remover.

k.

Using clean 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. Ensurethat the tissue is completely covered by FFPE Mounting Medium and the coverslip.
To ensure good image quality, IMMEDIATELY proceed to H&E imaging.

H&E Imaging
a.

Create a new folder in a fluorescent microscope-connected PC, name the folder withthe chip ID number and other essential information.
Use only letters, numbers, and underscores in the folder name. Special charactersand spaces are not allowed.
Example chip ID number: B00249A1b.

Turn on the fluorescence microscope, select the transmitted fluorescence (color)mode and choose the BF-Epi channel. Push the color/black and white slider into themachine and insert the light source diffuser.

c.

Place 1-2 μL of water on the imaging platform first, and then transfer and place theStereo-seq Chip Slide onto the water drop. Water surface tension will grab onto theslide and adhere it onto the imaging platform.

d. Position the chip directly under the objective lens and adjust the appropriate

Brightness, Gain, and Exposure parameters.

e.

Adjust the coarse adjustment knob until the tissue appears within focus, and thenuse the fine adjustment knob to further fine-tune the focus until the tissue and cell
contours can be clearly visualized.

f.

Move to the tissue area first, focus on the tissue, determine whether the color of thetissue area is normal. If the color appears to be off, move the focus to a blank areaon the chip to adjust the white balance first.

g.

Box-select the chip area to fully enclose the chip boundaries (which should beslightly larger than the chip), and scan the map under the 4X objective lens (if the
microscope does not have a map scanning function, skip to the next step).

h.

After scanning is completed, adjust the objective lens to 10X, then adjust theparameters such as Brightness (usually around 30, [0, 225]), Gain (set to theminimum), and Exposure (be careful not to overexpose). The track lines and thetissue morphology should be clear.

17

3. FFPE STANDARD OPERATING PROCEDURE

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manuali.

Shift the focus to a blank area on the chip, and perform background balancing.
Firstly adjust the focus to find a chip field of view without any tissue coverage or
obvious impurities. Adjust the focusing knob to zoom out for defocusing. If there aresmall impurities in the previously selected area, they should be barely visible after
defocusing. Finally, select "Background balancing".

j.

Further adjust the box-selected area and the edge of the chip according to themap, move to the tissue area first, then fine-tune the focus until the tissue and cell
contour can be clearly visualized.

k.

Select the region of interest within the tissue area, then manually select andestablish the model focal points. It is recommended that you first establish 3-5
model points in the blank areas of the chip (four corners of the chip).

l.

After establishing the model focal points in blank areas, focus on the tissue area toestablish the model focal point. It is suggested that you establish 4-6 model points indifferent tissue areas. When the model focal points have been established, perform
a full scan using a 10X lens.
If multiple chips need to be imaged, image with the same background balancing
parameters.
If the imaging is not satisfactory, retake the image.

m. Save the original tile (FOV) images, files, and stitched images.
n.

Open the ImageMap software and the Image Quality Control functional module
within the software. Upload your H&E-stained image and run Image QC according to
the instructions in the StereoMap User Manual within the software.

If the image fails to pass Image Quality Control, continue with the experimental
procedures and perform optimal image analysis later under the guidance of yourlocal Field Application Scientist.

Decolorization and Decrosslinking for H&E-stained Tissuea.

After H&E imaging, immerse the coverslip-mounted Stereo-seq Chip Slide in astaining jar (alternatively, use a slide container or a 50 mL centrifuge tube) filled withat least 30mL of 0.1X SSC, allow the coverslip to naturally detach from the tissuemounted chip, and gently rinse the Stereo-seq Chip Slide up and down to clean thechip surface.

b.

Take out the Stereo-seq Chip Slide and wipe off the excess solution from around andthe back of the slide with dust-free paper without touching the chip. Make sure there
is no liquid residue around the chip. The chip does not need to be air-dried.

c.

Assemble the Cassette and Gasket then place the Stereo-seq Chip Slide in the
Cassette according to the guide written in Appendix I: Stereo-seq Slide Cassette
Assembly. It is recommended that you practice with a regular blank glass slide.

d. Place the PCR Adaptor in the PCR thermal cycler and set the program as follows:18

3. FFPE STANDARD OPERATING PROCEDURE

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manual

Table 3-2 Decolorization and decrosslinking incubation program for H&E-stained tissue
Program selection: Incubate

Temperature

Time(Heated lid) 85°C

on

85°C

∞

85°C

20 min

30°C

5 min

30°C

∞

95°C

30 min

4°C

∞

e.

Steps

H&E Decolorization

H&E Decrosslinking

Grip along the Stereo-seq Cassette to make sure the Stereo-seq Chip Slide has been
locked in place.
Do not touch the front of the chip while assembling the Stereo-seq Slide Cassette.

f.

Add 400 μL TE Buffer (pH 9.0) that has been equilibrated to room temperature into
the well of the Stereo-seq Slide Cassette. Incubate at room temperature for 1 min,then discard the TE Buffer using a pipette.

g.

Add another 400 μL TE Buffer (pH 9.0) into the well of the Stereo-seq Slide Cassette.
Apply sealing tape to the Stereo-seq Slide Cassette and make sure it is sealed tightly.

h.

When the PCR thermal cycler reaches 85°C (Time = ∞), place the Stereo-seq Chip
Slide on the PCR Adaptor. Select Edit, and then select Next Step to skip the 85°C
(Time = ∞) step.
Do not preheat the TE Buffer, which may cause the tissue section to detach fromthe chip.

i.

Incubate the Stereo-seq Slide Cassette at 85°C for 20 min followed by 30°C for 5 min.

j.

When the PCR thermal cycler reaches 30°C (Time = ∞), remove the Stereo-seq Slide
Cassette from the PCR Adaptor, peel off the sealing tape, discard the TE Buffer (pH9.0) using a pipette, and proceed to the decrosslinking steps.

k.

Add 200 μL FFPE Decrosslinking Reagent into the well of the Stereo-seq Slide
Cassette. Incubate at room temperature for 30 sec ~ 1 min, then discard the liquidusing a pipette.

l.

Add another 400 μL FFPE Decrosslinking Reagent into the well of the Stereo-seq
Slide Cassette. Apply sealing tape to the Stereo-seq Slide Cassette and make sure it
is sealed tightly.

m. Place the Stereo-seq Slide Cassette on the PCR Adaptor in the PCR thermal cycler.

Select Edit, and then select Next Step to skip the 30°C (Time = ∞) step.

n.

Incubate at 95°C for 30min. Meanwhile, prepare about 30 mL of methanol in astaining jar (alternatively, use a slide container or a 50 mL centrifuge tube), close thelid, and pre-cool it for 5-30 min at -20°C.
19

3. FFPE STANDARD OPERATING PROCEDURE

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manualo.

When incubation is completed, carefully transfer the Stereo-seq Slide Cassette to
the nearest bench and peel off the sealing tape. Discard the FFPE Decrosslinking
Reagent with a pipette.

p.

Remove the slide from the Stereo-seq Slide Cassette according to the instructions inAppendix I: Stereo-seq Slide Cassette Assembly, discard the cassette and gasket,
then immediately proceed to the fixation step (Section 3.5).

The cassette and gasket may deform after heating to 95°C. DO NOT reuse thecassette and gasket. Discard them after this step. Use a new cassette and a newgasket in the subsequent steps.

3.4. Fluorescent Staining, Imaging and Decrosslinking
Stereo-seq Transcriptomics for FFPE workflow offers two staining options: H&E
staining or nuclei staining using ssDNA. Please choose one of the staining methods
and proceed to section 3.3 for the H&E staining workflow or section 3.4 for the
fluorescent staining workflow.

Reagent Required
for Section 3.3

Purpose

Preparation

5X SSC

Washing

Dilute 12.5 mL 20X SSC with 37.5 mL
ddH2O in a staining jar (alternatively,
use a slide container or a 50 mL
centrifuge tube) and mix well.

FFPE Mounting Medium

Take it out of -20°C in advance andChip Mounting equilibrate to room temperature until
it thaws.

0.1X SSC

Washing andcoverslip
detachment

TE Buffer (pH9.0)

FFPE Decrosslinking Reagent

Prepare 30~40 mL 0.1X SSC in astaining jar (alternatively, use a slide
container or a 50 mL centrifuge tube).

Take it out of 4°C in advance andH&E
decolorization equilibrate to room temperature atleast 5 min prior to use.
Take it out of -20°C in advance,
Decrosslinking equilibrate to room temperature until
it thaws, then mix well to ensure thatno white precipitates are visible.

If white precipitates are visible in the reagent, dissolve them by heating the bufferat 55 °C and equilibrate to room temperature before mixing.

20

3. FFPE STANDARD OPERATING PROCEDURE

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manual

Fluorescent Staininga.

Prepare fluorescent staining solution according to Table 3-3 and store it in the dark.
Table 3-3 Tissue fluorescent staining solution

Components

1X ( µL)

2X + 10% ( µL)

3X + 10% ( µL)

4X + 10% ( µL)

5X SSC

189

207.9

311.9

415.8

Qubit ssDNA Reagent 1

1.1

1.6

2.2

RI

10

11

16.5

22

Total

200

220

330

440

b.

Add 150 μL of tissue fluorescent staining solution per chip by first pipetting one
droplet at each corner of the chip and then adding the rest of the staining solution
to the middle to merge all droplets.
Ensure that the chip is completely covered by tissue fluorescent staining solution.

c.

Incubate the chip for 5 min at room temperature in the dark.

d. Gently remove the staining solution from the corner of the chip using a pipette.
e.

Add 150 μL of 0.1X SSC per chip then discard using a pipette.

f.

Repeat step e.

g.

Pipette 5 µL FFPE Mounting Medium onto the center of the tissue on the chip
without introducing air bubbles.
Make sure the coverslip is clean without any dust or debris. To clean the coverslip,
wipe it with an alcohol swab or use a power dust remover.

h.

Using clean 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. Ensurethat the tissue is completely covered by FFPE Mounting Medium and the coverslip.
To avoid fluorescent bleaching, IMMEDIATELY proceed to fluorescent imaging.

Fluorescent Imaging
a.

Create a new folder in a fluorescent microscope-connected PC, and name it with thechip ID number and other essential information.
Use only letters, numbers, and underscores in the folder name. Special charactersand spaces are not allowed.
Example chip ID number: B00249A1b.

Turn on the fluorescence microscope and set the epi-fluorescence channel to FITC.

c.

Place 1-2 μL of water on the imaging platform first, then transfer and place theStereo-seq Chip Slide onto the water drop. Water surface tension will grab onto the
Slide and adhere it to the imaging platform.

21

3. FFPE STANDARD OPERATING PROCEDURE

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manuald. Remove the light shield, switch to 4X objective lens, select the FITC channel andselect the chip area of interest. Adjust Brightness and Gain. The specific parameters
will vary among different microscopes, as long as the tissue can be imaged
clearly. However, the light intensity should be kept in the lower range to prevent
fluorescence quenching.

e.

Box-select the chip area to fully enclose the chip boundaries (should be slightly
larger than the chip), and scan the map under the 4X objective lens (if the
microscope does not have map scanning function, skip to the next step).

f.

Shift the focus to a blank area on the chip, finely adjust the focus until the tracklines
on the chip can be clearly visualized, and then manually select and establish themodel focal points. It is recommended that you first establish 3-5 model points inthe blank areas of the chip (four corners of the chip).

g.

After establishing the model focal points in blank areas, focus on the tissue area toestablish the model focal point. It is recommended that you establish 3-5 model
points in different tissue areas. When the model focal points have been established,perform a full scan using a 10X lens.

h.

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

i.

Open the ImageMap software and the Image Quality Control functional module
within the software. Upload your ssDNA-stained image and run Image QC according
to the instructions in the StereoMap User Manual within the software.
The captured nuclei-stained image needs to pass Image QC before further image
analysis (image “register”) can be performed in Stereo-seq Analysis Workflow (SAW)
pipelines.
If Image QC fails, continue with the experiment procedures and perform optimal
image analysis later under the guidance of your local Field Application Scientist.

Decrosslinking for ssDNA-stained tissuea.

Equilibrate FFPE Decrosslinking Reagent to room temperature in advance.

b.

After fluorescent imaging, immerse the coverslip-mounted Stereo-seq Chip Slide in astaining jar (alternatively, use a slide container or a 50 mL centrifuge tube) filled withat least 30mL of 0.1X SSC, allow the coverslip to naturally detach from the tissuemounted chip, and gently rinse the Stereo-seq Chip Slide up and down to clean thechip surface.
22

3. FFPE STANDARD OPERATING PROCEDURE

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manualc.

Take out the Stereo-seq Chip Slide and wipe off the excess solution from around andthe back of the slide with dust-free paper without touching the chip. Make sure there
is no liquid residue around the chip. The chip does not need to be air-dried.

d. Assemble the Cassette and Gasket then place the Stereo-seq Chip Slide in the

Cassette according to the guide written in Appendix I: Stereo-seq Slide Cassette
Assembly. It is recommended that you practice with a regular blank glass slide.

e.

Grip along the Stereo-seq Cassette to make sure the Stereo-seq Chip Slide has been
locked in place.
Make sure not to touch the front-side of the chip while assembling the Stereo- seq
Slide Cassette.
Place the PCR Adaptor in the PCR thermal cycler and set the program as follows:f.

Table 3-4 Decrossinglinking incubation program for ssDNA-stained tissue
Program selection: Incubate

Temperature

Time(Heated lid) 85°C

on

30°C

∞

95°C

30 min4°C∞g.

Add 400 μL FFPE Decrosslinking Reagent into the well of the Stereo-seq Slide
Cassette. Apply sealing tape to the Stereo-seq Slide Cassette and make sure it issealed tightly.

h.

Place the Stereo-seq Slide Cassette on the PCR Adaptor in the PCR thermal cycler.
Select Edit, and then select Next Step to skip the 30°C (Time = ∞) step.

i.

Incubate at 95°C for 30min. Meanwhile, prepare about 30 mL of methanol in astaining jar (alternatively, use a slide container or a 50 mL centrifuge tube), close thelid,and pre-cool it for 5-30 min at -20°C.

j.

When decrosslinking is completed, carefully transfer the Stereo-seq Slide Cassetteto the nearest bench and peel off the sealing tape. Discard the FFPE Decrosslinking
Reagent with a pipette.

k.

Remove the slide from the Stereo-seq Slide Cassette according to instructions inAppendix I: Stereo-seq Slide Cassette Assembly, discard the cassette and gasket,
then immediately proceed to the fixation step (Section 3.5).

The cassette and gasket may deform after heating to 95°C. DO NOT reuse thecassette and gasket. Discard them after this step. Use a new cassette and a newgasket in the subsequent steps.

23

3. FFPE STANDARD OPERATING PROCEDURE

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manual

3.5. Fixation
Reagent Required
for Section 3.5

Purpose

Preparation

MethanolFixation

Pre-cooled for 5-30 min at -20°C.

a.

Equilibrate the Stereo-seq Chip Slide to room temperature for 1 min, then immerse
the tissue-mounted Stereo-seq Chip Slide in the pre-cooled methanol for a 20 min
fixation at -20°C. Make sure that the entire tissue section is completely submerged.

b.

After fixation is completed, move the container to a sterile fume hood.

c.

Take out the Stereo-seq Chip Slide and wipe off excess methanol from around andthe back of the slide with dust-free paper without touching the chips. Make surethere is no methanol residue between chips.

d. Place the Stereo-seq Chip Slide on a slide staining rack and leave it in the fume hoodfor 4-6 min to let the methanol fully evaporate.

e.

When the methanol is fully evaporated, transfer the Stereo-seq Chip Slide onto a flatand clean bench.

f.

Assemble the Stereo-seq Slide Cassette with a new cassette and gasket.

g.

g. Grip along the Stereo-seq Cassette to make sure the Stereo-seq Chip Slide has
been locked in place.
Do not touch the front of the chip while assembling the Stereo-seq Slide Cassette.

24

3. FFPE STANDARD OPERATING PROCEDURE

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manual

3.6. Tissue Permeabilization
Reagent Required
for Section 3.6

Purpose

Preparation

0.01N HCl

Permeabilization

Prepare at least 1 mL of 0.01N HCl
per sample. Configure HCl to 0.01N.
Measure and make sure the pH = 2.

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 the experiments.
10X Permeabilization
Reagent Stock Solution

Add 1 mL of freshly prepared 0.01N
HCl to dissolve PR Enzyme (red cap, in
powder form), and thoroughly mix thereagent by pipetting.

Permeabilization

Do not vortex the permeabilization enzyme. Mix by pipette before using. Aliquot
this 10X stock solution to avoid freeze-thaw cycles.
1X Permeabilization
Reagent Solution

Permeabilization

Make 1X PR solution (200 μL / chip)by diluting 10X PR stock solution with
0.01N HCl.

0.1X SSC

Dilution

Add 10 μL 20X SSC into 1990 μL
nuclease-free water and mix well.

a.

Set aside the 2 mL of 0.01N HCl and 1X Permeabilization Reagent Solution you
prepared in 3.1 Experiment Preparation.

b.

Make sure your PCR thermal cycler has been switched on and set to 37°C with theheated lid set to 42°C. Place the PCR Adaptor in the PCR thermal cycler and set the
program as follows:
Table 3-5 Tissue permeabilization incubation program

Temperature

Time(Heated lid) 42°C

on

37°C

∞

37°C

30 min4°C∞c.

Add 200 μL of 1X Permeabilization Reagent Solution 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. Apply sealing tape to the Stereo-seq Slide Cassetteand make sure it is sealed tightly.
Make sure the chip is completely covered with 1X Permeabilization Reagent Solution.
25

3. FFPE STANDARD OPERATING PROCEDURE

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manuald. Thaw FFPE RT Oligo and FFPE Dimer on ice.
e.

Place the Stereo-seq Slide Cassette in the 37°C PCR thermal cycler and let the chipincubate inside the PCR thermal cycler at 37°C for 30 min.

f.

While waiting for permeabilization to be completed, prepare FFPE MIX Solution
according to Table 3-6 and leave it on ice until use. 【PREPARE AHEAD】
Table 3-6 FFPE MIX Solution
Components

1X ( µL)

2X + 10% ( µL)

3X + 10% ( µL)

4X + 10% ( µL)

FFPE RT Buffer Mix

158

347.6

521.4

695.2

FFPE RT Enzyme Mix

30

66

99

132

FFPE RT Oligo

10

22

33

44

FFPE Dimer

2

4.4

6.6

8.8

Total

200

440

660

880

g.

When incubation is completed, remove the Stereo-seq Slide Cassette from the PCR
Adaptor (37°C).

h.

Remove 1X Permeabilization Reagent Solution with a pipette from the corner of eachwell without touching the chip surface.

i.

Add 200 μL of 0.1X SSC (with 5% RI) per chip from the corner of each well.

j.

Remove 0.1X SSC (with 5% RI) with a pipette from the corner of each well without
touching the chip surface.

3.7. FFPE MIX Reactiona.

Place the PCR Adaptor in the PCR thermal cycler in advance. Set the temperature to
42°C and the heated lid to 45°C according to the following program:

Program selection: Incubate
Table 3-7 FFPE MIX hybridization incubation program

Temperature

Time(Heated lid) 45°C

on

42°C

∞

26

3. FFPE STANDARD OPERATING PROCEDURE

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manualb.

Gently add 200 μL of FFPE MIX Solution per chip along the side of each well,ensuring that the well surface is uniformly covered with FFPE MIX Solution. Applysealing tape to the Stereo-seq Slide Cassette and make sure it is sealed tightly.

c.

Place the Stereo-seq Slide Cassette in the 42°C PCR thermal cycler and let the chipincubate inside the PCR thermal cycler at 42°C for 5 hr or longer (no longer than 24
hr).

3.8. cDNA Release and Collection
PrepareReagent

Preparation Steps

Maintenance

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 precipitates are visible in the buffer, dissolve them by heating the bufferat 55 °C again and equilibrate to room temperature before mixing.
a.

Remove the Stereo-seq Slide Cassette from the PCR Adaptor, discard FFPE MIX
Solution, then wash one time with 200 μL 0.1X SSC per chip.

b.

Prepare the cDNA Release Mix according to Table 3-8 and maintain the mix at room
temperature.
Table 3-8 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

Componentsc.

1X ( µL)

Place the PCR Adaptor in the PCR thermal cycler in advance. Set the temperature to
55°C and the heated lid to 60°C according to the following program:
Table 3-9 cDNA Release Incubation Program

Temperature

Time(Heated lid) 60°C

on

55°C

5 hr - 24 hr

55°C

∞

27

3. FFPE STANDARD OPERATING PROCEDURE

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manuald. Add 400 μL of cDNA Release Mix per chip into each well of the Stereo-seq Slide

Cassette.

e.

e. Apply sealing tape to the Stereo-seq Slide Cassette and make sure it is sealed
tightly. Incubate the Stereo-seq Slide Cassette at 55°C for 5 hr or longer (no longer
than 24 hr).

Stop Point:
cDNA collection step may be left overnight. If it is left overnight, make sure the Stereoseq Slide Cassette is sealed tightly with the sealing tape.
f.

Before collecting the eluted cDNA, pre-heat 500 μL of nuclease-free water per chip at
55°C for ≥ 30 min.

g.

g. After the reaction, completely collect the cDNA Release Mix from each well into a
new 2.0 mL centrifuge tube.

h.

h. Add 350 μL of pre-heated nuclease-free water per chip into each well. Pipette up
and down to wash the chip surface thoroughly and then collect it into the same 2.0
mL centrifuge tube with the cDNA Release Mix.
Collect as much volume as possible to retrieve enough cDNA from the chip. cDNA
Release Mix should be about 400 μL after incubation (the volume might be lessthan 400 μL). You must combine the collected cDNA Release Mix with the 350 μL
nuclease-free water before proceeding to the next step.
The Stereo-seq Chip Slide may be discarded. Ensure that all of the chip ID numbers
have been recorded as required for downstream analysis.

3.9. 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 that the beads are thoroughly mixed every
time before use.

•

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

28

3. FFPE STANDARD OPERATING PROCEDURE

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manual

Operation Notes
1. In the magnetic separation step, 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 2-3
μL in the tube to avoid the pipette from direct 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.

29

3. FFPE STANDARD OPERATING PROCEDURE

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manual

4. After the second washing of beads with ethanol, try to remove all of the liquid
within the tube. You may centrifuge briefly to accumulate any remaining liquid
at the bottom of the tube, then separate the beads magnetically, and remove theremaining liquid by using 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 level. 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 prevent the pipette tip from directly contacting thebeads, always collect the eluate in 2 μL less than the initial volume of TE Buffer used
for the elution.

30

3. FFPE STANDARD OPERATING PROCEDURE

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE 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.

If white precipitates are visible in the collected cDNA, dissolve them by heating at
55°C and equilibrate to room temperature before proceeding to the purification
step. 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 (about 750 μL) with the beads in a ratio of 1 : 1. Vortex themix 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 remove and discard the supernatant with a pipette (if foam is visible on
the cap, discard it with a pipette).
4) Keep the tube on the magnetic separation rack and add 1.5 mL of freshly prepared
80% ethanol. 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 visible on
the cap, clean the cap with 80% ethanol).
5) Repeat step 4.
6) Keep the tube on the magnetic rack and open the lid to air-dry the beads at room
temperature until no wetness (reflectiveness) or crack is visible. Drying times willvary but will take approximately 5-15 min.
7) Add 44 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 (~ 42 μL cDNA) into a new 0.2 mL PCR tube.

31

3. FFPE STANDARD OPERATING PROCEDURE

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manual

If the volume of collected eluted cDNA is less than 42 μL, top it up with nuclease-freewater.

c.

Store the beads with 40 μL of nuclease-free water at 4°C after collecting theeluted cDNA untill your cDNA final product has passed QC.
d. Prepare PCR Mix according to Table 3-10. The total volume for the PCR reaction is

100 μL.

Table 3-10 PCR Mix
1X ( µL)

2X + 10% ( µL)

3X + 10% ( µL)

4X + 10% ( µL)

50

110

165

220

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 100

Components
cDNA Amplification
Mix
FFPE cDNA Primers
Mixe.

Mix gently and short spin before placing the reaction tube in a PCR thermal cycler.
Amplify the eluted cDNA based on the PCR program shown in Table 3-11.
Table 3-11 PCR program for amplification (for 100 μL)

f.

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

Prepare Qubit dsDNA Mix and record the concentration of PCR product according toTable 3-12.
Table 3-12 Qubit dsDNA Mix
Components

1X ( µL)

Invitrogen™ Qubit dsDNA HS Buffer

198

Qubit dsDNA HS Reagent 200X

1PCR Product

1

Total

200

32

3. FFPE STANDARD OPERATING PROCEDURE

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manualg.

Vortex the mix, then take 1 μL of the PCR product and measure its concentration
using Qubit dsDNA HS Kit. The DNA concentration is usually greater than 10 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 PCR product (100 μL) with the beads in a ratio of 1 : 1. Vortex the mix thenincubate 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 remove and discard the supernatant with a pipette (if foam is visible on
the cap, discard it with a pipette).
4) Keep the tube on the magnetic separation rack and add 200 μL of freshly
prepared 80% ethanol. 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 visible on
the cap, clean the cap with 80% ethanol).
5) Repeat step 4.
6) Keep the tube on the magnetic rack, and open the lid to air-dry the beads at room
temperature until no wetness (reflectiveness) or cracks are visible. Drying times willvary but will take approximately 5-8 min.
6) Add 42 μL of TE Buffer (pH 8.0) to the dried beads. Mix the beads and TE Buffer byvortexing. Incubate at room temperature for 5 min. Spin down briefly and place thesample tube onto a magnetic separation rack for 3-5 min until the liquid becomes
clear. Transfer the supernatant (~ 40 μL) into a new 1.5mL PCR tube.

Stop Point:
The purified cDNA 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 until 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.

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 at about
150- 350 bp (Figure 1), and a yield that is higher than 300 ng.

33

3. FFPE STANDARD OPERATING PROCEDURE

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manual

DV200 30%~50%

DV200 50%~70%

DV200 >70%
Figure 1. Representative Agilent Bioanalyzer 2100 analysis resultsof PCR amplified cDNA samples with different DV200 score34

3. FFPE STANDARD OPERATING PROCEDURE

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manual

Appendix I: Stereo-seq Slide Cassette Assembly

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

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

a.

c.

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

b.

If necessary, use a power dust
remover to blow any debris off the gasket.

d.

Press down on the gasket.

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.

35

1. INTRODUCTION

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manual

Make sure 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 cassettewith both middle fingers. Place your left
thumb between tab 1 and tab 2 and yourright thumb between tab 3 and tab 4.

g.

Press down evenly on the upper side
(A side) of the slide (near the edge) andthen simultaneously press down the topedge firmly with both index fingers to clip
the slide in place until you hear a clicking
sound.

h.

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

i.

j.

Recheck the Stereo-seq Slide Cassette and verify that the slide is clipped in place.

36

1. INTRODUCTION

Stereo-seq TRANSCRIPTOMICS SET FOR FFPE User Manual

Stereo-seq Slide Cassette Removal
To release the slide from the tabs, first flip the cassette over, and then as you gently
support the back of the Stereo-seq Chip Slide with both thumbs to prevent the Stereoseq Chip Slide from falling, firmly press the upper side down.

a.

b.

Lift the Stereo-seq Chip Slide from the side showing the engraved label.

37

1. INTRODUCTION