Stereo-seq Sample Preparation,
Sectioning and Mounting Guide
for Fresh Frozen Samples on
Stereo-seq Chip Slides
USER MANUAL

STUM_SP001

TABLE OF CONTENTS
CHAPTER 1: INTRODUCTION
1.1. Introduction							1CHAPTER 2: SAMPLE PREPARATION
2.1. Sample Requirements for Fresh Frozen Tissue		2.2. Sample Embedding for FF and PFA Samples		2.3. Sample Storage and Transportation			

3
4
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CHAPTER 3: CRYOSECTION PREPARATION
3.1. Stereo-seq Chip Slides					3.2. Cryosection Preparation					3.3. Tissue Mounting			
			

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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 Sample Preparation, Sectioning and Mounting Guide for Fresh Frozen Samples on Stereo-seq Chip Slides User Manual

1.1. Introduction
The STOmics Stereo-seq Transcriptomics Set is intended for generating a spatiallyresolved 3’ mRNA library from biological tissue sections and a Stereo-seq Chip
Slide with intact tissue sections as input. Prior to the Stereo-seq Transcriptomics
workflow, optimal permeabilization time needs to be determined using the Stereo-seq
Permeabilization Set for fresh frozen tissues. This guide provides general guidelines on
how to properly perform tissue embedding, sectioning, and mounting to better preserve
the morphological quality of the tissue sections and the integrity of mRNA transcripts.
The Stereo-seq Chip Slides prepared in this guideline are part of these products:
Stereo-seq Permeabilization Set for Chip-on-a-slide (211SP118)
Stereo-seq Transcriptomics Set for Chip-on-a-slide (211ST114)

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1. INTRODUCTION

CHAPTER 2
SAMPLE
PREPARATION

Stereo-seq Sample Preparation, Sectioning and Mounting Guide for Fresh Frozen Samples on Stereo-seq Chip Slides User Manual

2.1. Sample Requirements for Fresh Frozen Tissue
To prevent RNA degradation, we recommend performing tissue embedding within 30
min of harvesting.
The tissue size should not exceed 0.9 cm x 0.9 cm x 2 cm. The tissue section should
not exceed 80% area coverage of the chip.

Sample Types
This set of kits can be used for samples from all common animals, including but notlimited to human, monkey, and mouse.
For details, refer to the list: https://en.stomics.tech/resource/stomics-validatedtissue-list?lang=en#

Fresh Frozen Sample RNA Integrity Number (RIN) Value
It is recommended that you check the RNA quality (RIN value) of a tissue sample before
proceeding to the Stereo-seq experiment. Total RNA can be extracted from 10-20 slicesof 10 µm-thick tissue sections and stored at -20°C in a pre-cooled 1.5mL EP tube. Refer toFigure 1 for the peak of RNA RIN value in mouse brain tissue sections.
It is strongly recommended that you proceed only with tissue samples that have a
RIN value ≥7.

Figure 1. Example of RNA size distribution
and RIN value measurement of mouse brain
tissue sections3

2. SAMPLE PREPARATION

Stereo-seq Sample Preparation, Sectioning and Mounting Guide for Fresh Frozen Samples on Stereo-seq Chip Slides User Manual

2.2. Sample Embedding for FF Samples

For a demonstration video of tissue embedding, refer to the following link or scan the QR
code: https://en.stomics.tech/resources/videos
Preparation list:

Materials
Brand

Description

Quantity-

Crushed ice in a box

1-

Dry ice in a box

1-

Aluminum foil

1

BIOSHARP/Metal Coolbox/
BC032

Sealable plastic bag

1

Metal Block

1-

Sterile gauze

2

CorningCorning® 35 mm TC-treated Culture Dish
(353001)

1

Sakura/Base Molds/4583

OCT

1

Sakura/Base Molds/4162

Stainless-steel base mold A

1

Sakura/Base Molds/7055

Stainless-steel base mold B (slightly larger
than mold A)

1-

Blunt end forceps

1-

Syringe1-

Spatula1-

Scissors

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2. SAMPLE PREPARATION

Stereo-seq Sample Preparation, Sectioning and Mounting Guide for Fresh Frozen Samples on Stereo-seq Chip Slides User Manuala. Prepare these materials in advance:
a1. A box of crushed ice and pre-cool OCT on ice for 10 min in advance.
a2. 2 pieces of stainless-steel base molds slightly larger than the tissue of interest mold A and mold B (slightly larger than mold A).
a3. Add a few drops of pre-cooled OCT
in mold A until it reaches approximately
2/33. of the mold and pre-cool on ice for >10 min (remove introduced air bubbles
using a syringe).

a4. A petri dish filled with OCT andpre-cool the OCT on ice for > 10 min(remove introduced air bubbles usinga syringe).

a4.

a3.

a5. A box of dry ice.
a6. A metal block that has a flat surface to support the stainless-steel base moldwhen placed on dry ice. The size of the metal block should be larger than thestainless-steel base mold.
4.

a7. Place the metal block on dry ice
and pre-cool for > 5 min with the flat
surface facing up.
a7.

a8. Place mold B on dry ice and precool for > 5 min.

a8.

b. Upon harvesting within 30 min, usesterile gauze or dust-free paper to absorb
excess liquid on the tissue surface toprevent ice formation in later steps.

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2. SAMPLE PREPARATION

Stereo-seq Sample Preparation, Sectioning and Mounting Guide for Fresh Frozen Samples on Stereo-seq Chip Slides User Manualc. Place the tissue in pre-cooled OCT andwrap the tissue evenly with OCT using aspatula without introducing air bubbles.

d. Remove any air bubbles using asyringe.

e. Orient the tissue with the side
intended to be sectioned facing down
and then place it into mold A. Ensure thatthe tissue is at the bottom of mold A andfill the mold with chilled OCT, without
introducing bubbles, until the tissue isfully covered.

f. Place the tissue containing mold A
onto the metal block that was placed on
dry ice.

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2. SAMPLE PREPARATION

Stereo-seq Sample Preparation, Sectioning and Mounting Guide for Fresh Frozen Samples on Stereo-seq Chip Slides User Manualg. Use mold B as a lid with the opening
facing up, place it on top of mold A gently,
and then place a few dry ice cubes on top
of mold B. Ensure that the two stainlesssteel base molds can be covered withenough dry ice cubes.

h. After 5 min, remove mold B anddetermine if the OCT is completely
frozen and has turned opaque. If it is notcompletely frozen and opaque, repeat
step f.

i. If the tissue block has solidified andturned opaque, grip the two edges ofmold A and press down the edges todetach the tissue block from the mold.

j. Determine if the sectioning side of thetissue has been completely covered by
OCT. If not, place the tissue block on themetal block, sectioning side facing up,
add a few drops of the OCT, and then wait
until it solidifies and turns opaque.

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2. SAMPLE PREPARATION

Stereo-seq Sample Preparation, Sectioning and Mounting Guide for Fresh Frozen Samples on Stereo-seq Chip Slides User Manualk. Label the tissue block to mark theorientation of the tissue.

2.3. Sample Storage and Transportation
To store samples, wrap the tissue block with aluminum foil and keep it in a properlylabeled sealable plastic bag to prevent dehydration and damage, and then store it at-80°C. For transportation, ship samples on dry ice according to local policy.

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2. SAMPLE PREPARATIONCHAPTER 3CRYOSECTION PREPARATION

Stereo-seq Sample Preparation, Sectioning and Mounting Guide for Fresh Frozen Samples on Stereo-seq Chip Slides User Manual

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

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.

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3. CRYOSECTION PREPARATION

Stereo-seq Sample Preparation, Sectioning and Mounting Guide for Fresh Frozen Samples on Stereo-seq Chip Slides User Manual

3.2. Cryosection Preparation
Equipment

Preparation

Notes

Cryostat

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

The specimen disctemperature depends
on the tissue type.

a. Set a PCR Thermal Cycler to 37°C with the heated lid set to 42°C in advance with a
PCR adaptor.
b. Set the cryostat chamber temperature to -20°C and the pecimen disc temperature(object temperature) to -10°C~-15°C.
If the specimen disc is over-cooled, it could result in tissue section cracking
during sectioning, and sections will wrinkle when the disc temperature is toohigh. Optimal specimen disc temperature depends on the tissue type.
c. Place the forceps, brushes, and razor blades inside the chamber for pre-cooling.
d. Take the OCT-embedded tissue sample out of the -80°C freezer and place it in thecryostat chamber for 30 min to allow it to equilibrate to cryostat chamber temperature.
e. Remove the sample outer covers (aluminum foil) and trim the embedded tissue blockto the appropriate size (sectioning area smaller than 0.9 cm x 0.9 cm).
f. By using OCT, mount the embedded tissue block onto the specimen disc/holder of thecryostat chamber.
g. If necessary, trim the tissue block again to ensure a good fit between the tissue
section and the Stereo-seq Chip. The specimen is ready for cryosection.

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3. CRYOSECTION PREPARATION

Stereo-seq Sample Preparation, Sectioning and Mounting Guide for Fresh Frozen Samples on Stereo-seq Chip Slides User Manual

3.3. Tissue Mounting

For a demonstration video of tissue mounting onto the Stereo-seq Chip Slide, refer to thefollowing link or scan the QR code:
https://en.stomics.tech/resources/videos/list.html
a.

Take the Chip Slide out of the vacuum-sealed aluminum bag and record the Chip ID(SN)number that is on the back side of the slide. Do not touch the front side of thechip.

After opening the bag, confirm that all Stereo-seq Chip Slides in the slide
container are oriented with the front facing up. The front of a chip has a shiny
surface which contains DNB-probes for mRNA capture. DO NOT scratch thesurface.
b. Ensure that the PCR Thermal Cycler has been turned on and set to 37°C.
c.

Equilibrate Stereo-seq Chip Slide to room temperature for 1 min on the benchtop,
then rinse with 100 µL nuclease-free water twice with a pipette or rinse the slide up
and down twice in a 50 mL Corning tube with nuclease-free water.

Store unused slides in original packaging (first in the slide container andthen in the sealable aluminum bag) and keep sealed at -25°C ~ 8°C. KEEP THE
DESICCANT IN THE ALUMINUM BAG.
d. Remove excess water on the chip by blowing gently with a power dust remover(MATIN, M-6318). Wipe off excess water around the chip and on the slide with dustfree paper.

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3. CRYOSECTION PREPARATION

Stereo-seq Sample Preparation, Sectioning and Mounting Guide for Fresh Frozen Samples on Stereo-seq Chip Slides User Manuale.

The chip is ready for tissue mounting only when the chip is completely dry and there
are no wavy white stains.

f.

Prepare enough methanol in a 50 mL Corning tube or an empty slide container at
a volume that could submerge all of the chips on the slide. Immerse a regular glass
slide in the methanol containing tube to confirm that the volume is enough. Closethe lid and pre-cool the methanol for 5-30 min at -20°C.

g.

Place the tissue-mounted specimen disc/holder onto the cryostat head and adjust
the angle accordingly.

h.

Tissue mounting can be achieved using either the cold method (option A) or thewarm method (option B). We recommend practicing tissue mounting and section
placement on plain glass slides first.
A. Cold Method
1) Place Stereo-seq Chip Slide inside the chamber with the front facing up andpre-cool inside the cryostat chamber for 1-6 min.
Prolonged cooling for longer than 6 min may cause mist to form on the chipsurface.
2) Perform cryosection, then carefully flatten the tissue section by gently
touching the surrounding OCT with cryostat brushes. Place the tissue section
onto the chip center carefully with forceps and brushes. Ensure that the tissue
section is complete and without wrinkles.
3) Immediately pick up the Stereo-seq Chip Slide and place a finger on the backof the Stereo-seq Chip Slide directly under the chip for a few seconds to allow thesection to adhere to the chip.
4) Place the tissue-mounted Stereo-seq Chip Slide back inside the chamberand move on to the second tissue slicing and mounting. Continue transferring
sections on the remaining chips.
5) When all tissue mounting is completed, immediately dry the Stereo-seq Chip
Slide at 37°C in the PCR Thermal Cycler with PCR Adaptor for 5 min (without
heated-lid).
When performing cold mounting, mind the time interval between each tissue
section placement. Longer time intervals (>5 min) could result in tissue wrinkle
formation.
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3. CRYOSECTION PREPARATION

Stereo-seq Sample Preparation, Sectioning and Mounting Guide for Fresh Frozen Samples on Stereo-seq Chip Slides User Manual

B. Warm Method
1) Perform cryosection and obtain two or four consecutive tissue sections(depending on the number of chips on the Stereo-seq Chip Slide), carefully
flatten the tissue sections by gently touching the surrounding OCT with cryostat
brushes.
2) Move the tissue sections to the edge and place each tissue section at a distancegreater than the chip spacing on the Stereo-seq Chip Slide.
3) Flip the Stereo-seq Chip Slide and aim the tissue section within a chip area on
the Stereo-seq Chip Slide by gently touching the section with the front-side of thechip.
4) Repeat step 3) until all of the tissue sections have been mounted onto thechips of the Stereo-seq Chip Slide.
5) Turn the Stereo-seq Chip Slide over, and immediately dry it the PCR Thermal
Cycler at 37°C with PCR Adaptor front up for 5 min (without heated-lid).
If two different tissue blocks need to be cryosectioned and mounted onto thesame Stereo-seq Chip Slide, it is recommended that you first trim both tissue
blocks. Perform tissue sectioning and mounting for one tissue block first with thewarm method, and then place the tissue-mounted Stereo-seq Chip Slide in the
PCR Thermal Cycler for no longer than 5 min while preparing the second tissue
block. Perform tissue sectioning and mount the second tissue block using thewarm method, then place the tissue-mounted Stereo-seq Chip Slide in the PCR
Thermal Cycler to dry for 5 min.

Stop Point:•

After drying the tissue containing Stereo-seq Chip Slides in a PCR Thermal Cycler,
they can be stored in a slide container then transferred to a −80°C freezer on dry
ice.

•Store the sealed slide container containing Stereo-seq Chip Slides with tissue at−80°C for up to four weeks.

•

When retrieving Stereo-seq Chip Slides with tissue from the freezer, transfer out
the slide container on dry ice, and take out the tissue containing Stereo-seq Chip
Slides then immediately incubate at 37°C with PCR Adaptor for 5 min.

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3. CRYOSECTION PREPARATION

Stereo-seq Sample Preparation, Sectioning and Mounting Guide for Fresh Frozen Samples on Stereo-seq Chip Slides User Manual

Shipping Guidance for the Tissue Mounted Stereo-seq Chip Slide:•

After drying the tissue containing Stereo-seq Chip Slides on a PCR Thermal Cycler,transfer the Stereo-seq Chip Slide into a slide container then place it in a sealable
plastic bag. Place one desiccant pack per Stereo-seq Chip Slide into a sealable
plastic bag, push out as much air as possible and seal the bag tightly.

•

Prepare a styrofoam shipping container filled with dry ice. Allow 0.5 kg of dry ice
for every 2 hours in transport. For example, for a 3-day shipping duration, 0.5kg *12hr * 3 = 18kg of dry ice can be prepared.

•

Place the ziplock bag at the bottom layer of the shipping box. If necessary, use
sheets of bubble wrap to ensure the slide container remain in a vertical position.

•

Fill the empty space in the box with bubble wrap or paper. This will help prevent
shifting of the slide container when the ice dissipates.

•

Close the styrofoam lid. DO NOT tape the styrofoam lid to the box.

•

Secure the outer lid of the shipping box with tape. When using dry ice, it isrecommended that you leave an air gap when taping to ensure that carbon dioxide
can be released. This can prevent a buildup of pressure that could rupture thepackage.

•Label the shipping box with a dry ice sticker and then adhere the pre-printed label
showing both the recipient's address and the return address. Contact the recipientlab before shipping to ensure that the staff is prepared to receive the shipment.

•

When retrieving the Stereo-seq Chip Slides with tissue from the carrier, store
the Stereo-seq Chip Slides at −80°C (less than 4 weeks from mounting) until you
are ready to process the slide. Before processing, take out the tissue containing
Stereo-seq Chip Slides, immediately incubate at 37°C on the PCR Adaptor for 5min, and then proceed to tissue fixation.
Shipping guidance is based on STOmics in-house validation and general shipping
guidance provided by CDC www.cdc.gov.

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3. CRYOSECTION PREPARATION