Stereo-seq OMNI FFPE Library
Preparation
USER MANUAL
with Stereo-seq 16 Barcode Library Preparation Kit V1.0

Cat. No.: 111KL160 (16 RXNs)
Kit Version: V1.0
Manual Version: A

STUM-LP001

Stereo-seq OMNI FFPE Library Preparation User Manual

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

A
V1.0
Jul. 2024
Initial release2

Stereo-seq OMNI FFPE Library Preparation User Manual

Note: Please download the latest version of the manual and use it with thecorresponding Stereo-seq Transcriptomics Kit.
©2024 STOmics Tech Co., Ltd. at Shenzhen.
All rights reserved.
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3

Stereo-seq OMNI FFPE Library Preparation 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
1
1
2

CHAPTER 2: LIBRARY PREPARATION FOR FFPE SAMPLES
2.1. Experiment Preparation
5
2.2. cDNA Amplification and Purification cDNA
5
LIBRARY CONSTRUCTION FOR STEREO-SEQ FFPE
TRANSCRIPTOME LIBRARY

9Appendix A: PCR Barcode Primer Mix Use Rules

10

NOTE: Additional operation tips and guidance.
CAUTION: Proceed with extra care; improper handling or carelessness may cause
experimental failure or accidents.

4

CHAPTER 1
INTRODUCTION

Stereo-seq OMNI FFPE Library Preparation User Manual

1.1. Intended Use
Stereo-seq OMNI FFPE Library Preparation requires the usage of Stereo-seq 16 Barcode
Library Preparation Kit, which is designed for samples utilizing Stereo-seq technology,
including fresh frozen transcriptome cDNA samples, multi-protein antibody-derived
tags (ADT) samples, and FFPE transcriptome cDNA samples. It enables the addition ofsample barcodes and library construction, suitable for multi-sample mixed sequencing,supporting up to 16 samples for mixed sequencing.
All reagents provided within this kit have passed stringent quality control and functionalverification, ensuring performance stability and reproducibility of library construction,
as well as the uniformity and accuracy of sequencing data splitting.

1.2. Sequencing Guideline
Sequencing libraries produced via the Stereo-seq Transcriptomics Set require the
DNBSEQ sequencing platform For details, please refer to Library Construction for Stereoseq FFPE Transcriptome Library.
Necessary input parameters for Stereo-seq FFPE transcriptome libraries for the Stereoseq Analysis Workflow (SAW) bioinformatics pipelines:--kit-version= 'Stereo-seq N FFPE V1.0'--sequencing-type = 'PE75_25+59'

1.3. List of Kit Components
Each Stereo-seq 16 Barcode Library Preparation Kit consists of:Table 1-1

Stereo-seq 16 Barcode Library Preparation Kit V1.0

Cat. No.: 111KL160

Component

Reagent Cat. No. Cap Color

Quantity (tube)

KMB

1000047709

160 µL

×1

KME

1000047770

80 mg

×1

PCR Barcode Primer Mix1

1000043201

25 µL

×1

PCR Barcode Primer Mix2

1000043202

25 µL

×1

PCR Barcode Primer Mix3

1000043203

25 µL

×1

PCR Barcode Primer Mix4

1000043204

25 µL

×1

PCR Barcode Primer Mix5

1000043205

25 µL

×1

PCR Barcode Primer Mix6

1000043206

25 µL

×1

PCR Barcode Primer Mix7

1000043207

25 µL

×1

1

1. INTRODUCTION

Stereo-seq OMNI FFPE Library Preparation User Manual

Stereo-seq 16 Barcode Library Preparation Kit V1.0

Cat. No.: 111KL160

Component

Reagent Cat. No. Cap Color

Quantity (tube)

PCR Barcode Primer Mix8

1000043208

25 µL

×1

PCR Barcode Primer Mix9

1000043209

25 µL

×1

PCR Barcode Primer Mix10

1000043210

25 µL

×1

PCR Barcode Primer Mix11

1000043211

25 µL

×1

PCR Barcode Primer Mix12

1000043212

25 µL

×1

PCR Barcode Primer Mix13

1000043213

25 µL

×1

PCR Barcode Primer Mix14

1000043214

25 µL

×1

PCR Barcode Primer Mix15

1000043215

25 µL

×1

PCR Barcode Primer Mix16

1000043216

25 µL

×1

PCR Amplification Mix

1000043217

800 µL

×1

Storage Temperature:-25°C~-18°C

Transported
by cold chain

Expiration Date:refer to label

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

1.4. Additional Equipment and Materials
The table below lists equipment and materials needed for this protocol. Some common
laboratory equipment not named in Table 1-2 are expected to be accessible by the user,
for instance, an ice maker, biological safety cabinet, freezers, etc.
Table 1-2

Equipment
Brand

Description

Cat. No.

-

PCR thermocycler-

NEBNext®

Magnetic Separation Rack for <200 μL tubes S1515S

Thermo Fisher
Scientific
Agilent
Technologies™

Magnetic rack DynaMag™-2 for 1.5-2mL tubes 12321D
Q33216
Qubit™3 fluorometer(or similar)
Vortex mixer
Agilent 2100 bioanalyzer
2

G2939AA(or similar)
1. INTRODUCTION

Stereo-seq OMNI FFPE Library Preparation User Manual

Reagents
Brand
Ambion*Beckman Coulter*VAZYME
Invitrogen
Agilent
Technologies™

Description

Cat. No.

Nuclease-free water

AM9937

1X TE buffer, pH 8.0

AM9858

100% Ethanol (Analytical grade)-

AMPure® XP

A63882SPRIselect

B23317/B23318/
B23319VAHTS™ DNA Clean Beads

N411-02

Qubit dsDNA HS Assay Kit

Q32854

Qubit ssDNA Assay Kit

Q10212High sensitivity DNA kit

5067-4626

Description

Cat. No.

1.5 mL centrifuge tubes

MCT-150-A

0.2 mL PCR tubes*

PCR-02-C

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

1,000 μL filtered tips

TF-1000-L-R-S

200 μL filtered tips

TF-200-L-R-S

100 μL filtered tips

TF-100-R-S

10 μL filtered tips

TXLF-10-L-R-S

0.5 mL thin wall PCR tubes

PCR-05-C

Qubit Assay Tubes

Q32856

Consumables
Brand

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

3

1. INTRODUCTION

CHAPTER 2
LIBRARY PREPARATION FOR
FFPE SAMPLES

Stereo-seq OMNI FFPE Library Preparation 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

80% Ethanol

Dilute 100% ethanol to 80%

Roomtemperature up to
1 day

Magnetic beads

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

4°C

2.2 cDNA Amplification and Purification cDNAa.

Use 20 ng cDNA sample for the amplification reaction according to Table 3-1.
Table 3-1 Amplification Reaction Mix

Components

1X ( µL)

PCR Amplification Mix

50

PCR Barcode Primer Mix

25

cDNA Product

X

Nuclease-free water

25-X

Total

100

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

5

2. LIBRARY PREPARATION FOR FFPE SAMPLES

Stereo-seq OMNI FFPE Library Preparation User Manualb.

Program a thermocycler according to Table 3-2. When the module starts to heat up,
put the reaction tube into the thermocycler.
Table 3-2 Reaction program (100μL)

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

-

c.

8

Take 1 μL of the PCR product and use the Qubit dsDNA HS Kit to measure theconcentration. The concentration is usually more than 5 ng/μL.

d. Purify the above reaction products with 0.8x magnetic beads.

1) Mix the cDNA PCR product (100 µL) with beads in a ratio of 1 : 0.8. Vortex the
mixture then incubate it at room temperature for 10 min.
2) Spin down and place the sample tube onto a magnetic separation rack for 3-5
min until the liquid becomes clear. Carefully remove and discard the supernatantwith a pipette.
3) Add 200 μL of 80% ethanol for washing, using freshly prepared andequilibrated 80% ethanol at room temperature. During the washing process, thetube should always be placed on the magnetic rack, and the pipette tip should
operate on the wall of the tube away from the magnetic rack. Do not aspirate or
disturb the magnetic beads. Keep the tube on the magnetic rack.
4) Repeat step 3) one more time.
5) Keep the tube on the magnetic rack, and open the lid to air-dry the beads atroom temperature until no wetness (reflectiveness) is observed. Drying times willvary but will be approximately 5-8 min.
6) Add 42 μL of TE buffer to the dried beads. Mix the beads and TE buffer byvortexing. Incubate at room temperature for 5 min. Spin down briefly and place
the sample tube onto a magnetic separation rack for 3-5 min until the liquid
becomes clear. Transfer the supernatant to a new 1.5 mL tube.
e.

Take 1 μL of purified PCR product and measure the concentration with Qubit dsDNA
HS Kit. Use Bioanalyzer, Tapestation (Agilent Technologies), LabChip® GX, GXII, GX
Touch (PerkinElmer), Fragment Analyzer™ Advanced Analytical) or other equipment
based on the principle of electrophoretic separation to detect the fragment
distribution of the purified PCR products. The main peak of fragment distribution isaround 100-1000 bp (Figure1). Normally, the PCR yield is higher than 100 ng.

6

2. LIBRARY PREPARATION FOR FFPE SAMPLES

Stereo-seq OMNI FFPE Library Preparation User Manual

DV200 30%~50%

DV200 50%~70%

DV200 >70%
Figure 1. Agilent 2100 Bioanalyzer fragment size distribution of
the FFPE Libraries from samples with different DV200 score7

2. LIBRARY PREPARATION FOR FFPE SAMPLESCHAPTER 3
LIBRARY CONSTRUCTION FOR
Stereo-seq FFPE
TRANSCRIPTOME LIBRARY

Stereo-seq OMNI FFPE Library Preparation User Manual

The structure of the Stereo-seq OMNI FFPE library structure is shown in the figure below.
Refer to the user manual of DNBSEQ-G400RS Stereo-seq Visualization Reagent Set,
940-001886-00 (MGI)/940-001885-00 (CG) or DNBSEQ-T7RS Stereo-seq Visualization
Reagent Set, 940-001895-00 (MGI)/940-001889-00 (CG) for DNB preparation.
Recommended library input is 60ng.

Use the following parameters to perform the sequencing run:
• Choose paired-ended mode with 25 cycles of Read 1 and 59 cycles of Read 2 and an
additional 10 cycles of sample barcode.
Please read the corresponding user manual carefully before performing sequencing
and strictly follow the instructions. If you have any questions about sequencing, pleasecontact your local technical support for DNBSEQ platforms.

9

3. LIBRARY CONSTRUCTION

Stereo-seq OMNI FFPE Library Preparation User Manual

Appendix A: PCR Barcode Primer Mix Use Rules
The Stereo-seq 16 Barcode Library Preparation Kit V1.0 provides 16 types of PCR Barcode
Primer Mix, designed to meet the needs of high-throughput library construction for alarge number of samples and multiplex sequencing of multiple samples. This kit is basedon the design principle of base balance and has been tested by repeated experiments.
To ensure the best results, please read the following usage rules carefully.
Any sample with the same barcode cannot be sequenced in the same lane.
Based on the base balancing principle, the PCR Barcode Primer Mix should be used ingroups with the following grouping rules:
4 PCR Barcode Primer Mixes in groups: 1~4, 5~8, 9~12, 13~16, a total of 4 sets.
Make sure to centrifuge the PCR Barcode Primer Mix thoroughly before use. Gently
open the lid during use, to prevent the liquid from splashing and avoid crosscontamination. Pipetting to mix well and shortly centrifuge while using. Close thelid in time after use.
N types of PCR Barcode Primer Mix Method: take the same volume of each; mix
them and add the mixture to the sample.

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

Method 2

Method 3

Method 4

1

1 to 45 to 89 to 1213 to 162

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

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

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

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

3

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

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

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

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

4

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

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

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

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

5

Sample 1: 1
Sample 2: 2
Sample 3: 3
Sample 4: 4
Sample 5: selectany group from theremaining threegroupsSample 1: 5
Sample 2: 6
Sample 3: 7
Sample 4: 8
Sample 5: selectany group from theremaining threegroupsSample 1: 9
Sample 2: 10
Sample 3: 11
Sample 4: 12
Sample 5: selectany group from theremaining threegroupsSample 1: 13
Sample 2: 14
Sample 3: 15
Sample 4: 16
Sample 5: selectany group from theremaining threegroups10

Stereo-seq OMNI FFPE Library Preparation User ManualSample/lane Method 1

Method 2

Method 3

Method 4

6

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

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

8

Select any two groups from the four groups.

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

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

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

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

N= 9~16

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

When the library data amount requirements are different:
Libraries that require more than 20% data in a lane are required to use grouped PCR
Barcode Primer Mix
Example:If there are 9 samples pooled in a lane, and 1 of them requires 30% data, thefollowing scheme is required: if the other 8 samples each use PCR Barcode Primer
Mix 1~8, this sample should not use a single PCR Barcode Primer Mix, but use a nonduplicative and grouped PCR Barcode Primer Mix 9~12 or 13~16.
11