User Guide | CG000685 | Rev B

VisiumHD Spatial Gene Expression Reagent
Kits
For use with:
Visium HD Reagent Kit, Small, PN-1000668
Visium Human Transcriptome Probe Kit v2 - Small, PN-1000466
Visium Mouse Transcriptome Probe Kit v2 - Small, PN-1000667
Visium HD Cassettes, 6.5 mm, 4 rxns, PN-1000669
Visium HD Slide, 6.5 mm, 2 rxns, PN-1000670
Visium CytAssist Reagent Accessory Kit, PN-1000499
Dual Index Kit TS Set A, 96 rxns PN-1000251

Manufactured under license from Centrillion

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NoticesDocument Number
CG000685 | Rev B

Legal Notices© 2024 10x Genomics, Inc. (10x Genomics). All rights reserved. Duplication and/or reproduction of all or any
portion of this document without the express written consent of 10x Genomics, is strictly forbidden. Nothing
contained herein shall constitute any warranty, express or implied, as to the performance of any products
described herein. Any and all warranties applicable to any products are set forth in the applicable terms andconditions of sale accompanying the purchase of such product. 10x Genomics provides no warranty and hereby
disclaims any and all warranties as to the use of any third-party products or protocols described herein. The useof products described herein is subject to certain restrictions as set forth in the applicable terms and conditionsof sale accompanying the purchase of such product. A non-exhaustive list of 10x Genomics’ marks, many ofwhich are registered in the United States and other countries can be viewed at:www.10xgenomics.com/trademarks. 10x Genomics may refer to the products or services offered by othercompanies by their brand name or company name solely for clarity, and does not claim any rights in those thirdparty marks or names. 10x Genomics products may be covered by one or more of the patents as indicated at:www.10xgenomics.com/patents. The use of products described herein is subject to 10x Genomics Terms andConditions of Sale, available at www.10xgenomics.com/legal-notices, or such other terms that have been agreed
to in writing between 10x Genomics and user. All products and services described herein are intended FOR
RESEARCH USE ONLY and NOT FOR USE IN DIAGNOSTIC PROCEDURES.

Instrument & Licensed Software Updates Warranties
Updates to existing Instruments and Licensed Software may be required to enable customers to use new orexisting products.

Support
Email: support@10xgenomics.com
10x Genomics
6230 Stoneridge Mall Road
Pleasanton, CA

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Document Revision Summary
Document Number
CG000685

Title
Visium HD Spatial Gene Expression Reagent Kits User Guide

Revision
Rev B

Revision Date
September 10, 2024

Description of ChangeslAdded additional tips on ensuring Visium HD slide is dry to Visium HD Slide Handling on page 23.

lAdded new image demonstrating reagent removal to Reagent Addition to & Removal from Wells on page 25.

lAdded additional information on Visium CytAssist alignment guides in Instrument Loading Guidelines on
page 34.

l

Added Fresh Frozen and Fixed Frozen documentation to Workflow Overview on page 13.

lAdded Fresh Frozen and Fixed Frozen information to Sample Preparation on page 43.

l

Adjusted time a Visium HD Slide may remain in buffer prior to instrument run in 3.1 Visium HD Slide Washon page 59.

lAdded new visual overview in 4.1 CytAssist-Enabled Probe Release & Capture on page 65.

l

Added information on color balance to Sample Indices on page 97.

l

Added background on recommended sequencing depth in Sequencing Depth on page 90.

lAdded NovaSeq X plus to Illumina Sequencer Compatibility on page 97 and Sequencing Metrics on page 99.

lAdded note on consequences of not drying the Visium HD slide to 1. Bubbles Trapped During Visium
CytAssist Run on page 103.

l

Added information on high split-mapped or half-mapped reads in 5. High Split-Mapped or Half-Mapped
Reads on page 110.

l

Added information on data loss due to hydrogel damage in 6. Data Loss due to Hydrogel Damage on
page 111.

l

Added sequences to Oligonucleotide Sequences on page 122.

lUpdated for general minor consistency of language and terms throughout.

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Table of Contents
Contents
Introduction
Reagent Kits
10x Genomics Accessories
Third-Party Items
Workflow Overview
Protocol Steps & Timing
Stepwise Objectives
Visium HD Slides

8
11
12
13
14
15
16

Tips & Best Practices
General Reagent Handling
Visium Slide Storage
Pipette Calibration
Visium HD Slide HandlingTissue Slide Handling
Reagent Addition to & Removal from Wells
Visium Slide Seal Application & Removal
Slide Incubation Guidance
Cassette Incubation
10x Magnetic Separator
Magnetic Bead Cleanup StepsSPRIselect Cleanup & Size Selection
Sample Indices (i5/i7) in Sample Index PCR

23
23
23
23
24
25
27
28
29
30
30
31
31

Visium CytAssist
Instrument Orientation
Instrument Loading Guidelines

33
34

Sample Preparation & Staining Guidelines
Sample Preparation

43

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Table of Contents
Step 1: Probe Hybridization
1.0 Get Started
1.1 Probe Hybridization

46
47

Step 2: Probe Ligation
2.0 Get Started
2.1 Post-Hybridization Wash
2.2 Probe Ligation
2.3 Post-Ligation Wash

51
52
54
55

Step 3: Visium HD Slide Preparation
3.0 Get Started
3.1 Visium HD Slide Wash

58
59

Step 4: Probe Release & Extension
4.0 Get Started
4.1 CytAssist-Enabled Probe Release & Capture
4.2 Probe Extension
4.3 Probe Elution

63
65
75
76

Step 5: Pre-Amplification and SPRIselect
5.0 Get Started
5.1 Pre-Amplification
5.2 Pre-Amplification Cleanup - SPRIselect

78
79
80

Step 6: Visium HD Spatial Gene Expression – Probe-based Library
Construction
6.0 Get Started
6.1 Cycle Number Determination – qPCR
6.2 Sample Index PCR
6.3 Post-Sample Index PCR Cleanup – SPRIselect
6.4 Post-Library Construction QC

82
83
85
87
88

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Table of Contents
Sequencing
Sequencing Libraries
Sequencing Depth
Sequencing Type & Run Parameters
Illumina Sequencer Compatibility
Sample IndicesLibrary Loading
Sequencing Metrics

90
90
97
97
97
98
99

Troubleshooting
Before CytAssist Instrument Run
During CytAssist Instrument Run
After CytAssist Instrument Run
Issues Impacting Tissue Analysis

102
103
105
108

Appendix
HD Workflow Overview for Processing 4 Tissue Slides with 1 Visium CytAssist
Instrument
HD Workflow Overview for Processing 4 Tissue Slides with 2 Visium CytAssist
Instruments
Post Library Construction Quantification
LabChip Traces
Agilent TapeStation Traces
Oligonucleotide Sequences

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116
119
120
121
122

6

Introduction
Reagent Kits

8

10x Genomics Accessories

11

Third-Party Items

12

Workflow Overview

13

Protocol Steps & Timing

14

Stepwise Objectives

15

Visium HD Slides

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Reagent Kits
Components

Visium HD, Human
Transcriptome, 6.5 mm*

1000675

Visium HD Slide, 6.5 mm, 2
rxns

1000670

Visium Human Transcriptome
Probes v2 - small

1000466

Visium HD Reagents, small

1000668

Visium HD Cassettes, 6.5 mm,
4 rxns1000669

Visium HD Slide, 6.5 mm, 2
rxns

1000670

Visium Mouse Transcriptome
Probes v2 - small

1000667

Visium HD Reagents, small

1000668

Visium HD Cassettes, 6.5 mm,
4 rxns1000669

1000676

Tab 2 Text

Part Number

Visium HD Mouse,
Transcriptome, 6.5 mm*

Tab 1 Text

Component Part
Number

Reagent Kits

Tab 3 Text

*Also available in a pack of 4 as a 16 rxn kit.

Visium HD Slide, 6.5 mm, 2 rxns PN-1000670
Visium HD Slide, 6.5mm
2 rxns PN-1000670(store at −80°C)
PN

12000970

Tab 4 Text

Visium HD Slide, 6.5 mm#

Tab 5 Text
Tab 6 Text

Introduction

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Visium HD Cassettes*, 6.5 mm, 4 rxns PN-1000669#

PN

Visium Cassette 2-port gasket, 6.5 mm

23001831

Visium Cassette Bottom

2300183043001826

Movable Tissue Gasket 6.5 mm (preassembled with translator)

43001828

Movable Tissue Gasket Translator (preassembled with gasket)

4300192743001825

2

2000283

Tab 1 Text

Visium HD Cassettes, 6.5 mm
4 rxns PN-1000669(store at ambient temperature)
Visium 2-port Cassette S3, 6.5 mm

Visium Tissue Slide Cassette S3, 6.5 mm

Tissue Slide Cassette Bottom
Visium Slide Seals, 12 packTab 2 Text

Tissue Slide Cassette Top

Tab 3 Text

*The Visium HD Workflow is run with Visium Tissue Slide Cassettes S3 andVisium Cassettes S3. These are referred to as Tissue Slide Cassettes andVisium Cassettes in this document, respectively. Consult the Visium CassetteS3 Quick Reference Card (CG000730) for assembly and disassembly
information.

Visium HD Reagent Kit – Small, PN-1000668
Tab 4 Text

Visium HD Reagent Kit – Small
PN-1000668(store at −20°C)

Amp Mix B

12000567

Extension
Enzyme12000389

Extension Buffer

12000409

RNase Enzyme

1

3000605

2X RNase Buffer

12000411Perm Enzyme B

13000553

TS Primer Mix B

12000537

Decrosslinking
Buffer B

1

2001094

Tab 6 Text

PN

Tab 5 Text

Introduction#

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Visium Human Transcriptome Probe Kit v2 - Small, PN-1000466

FFPE Hyb Buffer

12000423

FFPE Post-Hyb
Wash Buffer

12000424

Human WT Probes
v2 – RHS

1

2000657

Human WT Probes
v2 – LHS

12000658

Probe Ligation
Enzyme12000425

2X Probe
Ligation Buffer

12000445

Post Ligation Wash
Buffer1

2000419

Tab 3 Text

PN

Tab 2 Text#

Tab 1 Text

Visium Human Transcriptome Probe Kit v2 Small
PN-1000466(store at −20°C)

Visium Mouse Transcriptome Probe Kit v2- Small, PN-1000667

FFPE Hyb Buffer

12000423

FFPE Post-Hyb
Wash Buffer

12000424

Mouse WT Probes
v2 – RHS

1

2000913

Mouse WT Probes
v2 – LHS

1

2000912

Probe Ligation
Enzyme12000425

2X Probe
Ligation Buffer

12000445

Post Ligation Wash
Buffer1

2000419

Tab 6 Text

PN

Tab 5 Text

Introduction#

Tab 4 Text

Visium Mouse Transcriptome Probe Kit v2 Small
PN-1000667(store at −20°C)

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Dual Index Kit TS Set A, 96 rxns PN-1000251

Dual Index Plate TS Set A

#

PN

1

3000511

Tab 1 Text

Dual Index Kit TS Set A
96 rxns PN-1000251(store at −20°C)

Tab 2 Text

10x Genomics Accessories
Product#

10x Magnetic Separator

1

Low Profile Thermocycler
Adapter2

Part Number (Kit)

1000499 (Visium CytAssist Reagent
Accessory Kit)

Part Number(Item)
230003 or
20004313000823

Tab 3 Text
Tab 4 Text
Tab 5 Text
Tab 6 Text

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Third-Party Items
Tab 1 Text

Successful execution of the Visium HD workflow requires third-party
reagents, kits, and equipment in addition to those provided by 10x Genomics.
All third-party reagents and consumables should be obtained prior to starting
this workflow.
Consult the Visium HD Spatial Gene Expression Protocol Planner (CG000698,
Rev B or later) for a list of the following third-party items:
Additional reagents, kits, and equipmentlTested pipette tipslTested thermal cyclerslTested blank slides

Tab 2 TextlTab 3 Text

10x Genomics has tested all items listed in the Protocol Planner. Unlessotherwise noted, 10x Genomics recommends using these items to ensure
optimal assay performance. Substituting materials may adversely affect assay
performance.

Tab 4 Text
Tab 5 Text
Tab 6 Text

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Workflow Overview
Sample Preparation

Visium HD FF Tissue
Preparation Handbook
Prepare Fresh Frozen tissue
blocks, section tissue ontoslides, stain, and image.
Demonstrated Protocol
CG000763

Visium HD FxF Tissue
Preparation Handbook
Prepare Fixed Frozen tissue
blocks, section tissue ontoslides, stain, and image.
Demonstrated Protocol
CG000764

Visium HD Spatial
Applications Imaging
Guidelines
Optimize imaging settings.
Technical Note CG000688

Visium Cassette S3 Quick
Reference Card
Practice cassette assemblyand disassembly.
Quick Reference Card
CG000730

Visium HD Spatial
Applications Protocol
Planner
Information on third-party
items.
Planner
CG000698

Tab 3 Text

Visium HD FFPE Tissue
Preparation Handbook
Prepare formalin fixed &paraffin embedded tissue
blocks, section tissue ontoslides, stain, and image.
Demonstrated Protocol
CG000684

Tab 2 Text

Before starting thisUser Guide, consult
these documents to
prepare samples.
Choose one handbook
for guidance on tissue
preparation andstaining.

Tab 1 Text

1

Tab 4 Text

Visium CytAssist
Accessory Kit Quick
Reference Card
Determine slide allowableareas.
Quick Reference Card
CG000548

Library Construction

Tab 5 Text

2

Visium HD Spatial Gene
Expression Reagent Kits
User Guide
Construct Visium HD Spatial
Gene Expression - Probebased Libraries.
User Guide CG000685

Introduction

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Tab 6 Text

Consult the 10x Genomics support website for additional documents

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Protocol Steps & Timing
TimingTab 1 Text

Steps

Stop & Store

Day 1
Step 1: Probe Hybridization (page 45)
1.1 Probe Hybridization (page 47)

Overnight

Day 2

2.1 Post-Hybridization Wash (page 52)

18 min2.2 Probe Ligation (page 54)

60 min2.3 Post-Ligation Wash (page 55)

12 minTab 2 Text

Step 2: Probe Ligation (page 50)

4°C ≤24 h

Step 3: Visium HD Slide Preparation (page 57)
20 minTab 3 Text

3.1 Visium HD Slide Wash (page 59)
4.1 CytAssist-Enabled Probe Release & Capture (page 65)
4.1 CytAssist-Enabled Probe Release & Capture (page 65)

60 min4.2 Probe Extension (page 75)

60 min4.3 Probe Elution (page 76)4.3 Probe Elution (page 76)

15 min4°C ≤24 h

Tab 4 Text

Step 5: Pre-Amplification and SPRIselect (page 77)
5.1 Pre-Amplification (page 79)

40 min5.2 Pre-Amplification Cleanup - SPRIselect (page 80)

30 min4°C ≤72 h or -20°C ≤4
weeks

Day 3

6.1 Cycle Number Determination – qPCR (page 83)

45 min6.2 Sample Index PCR (page 85)

40 min6.3 Post-Sample Index PCR Cleanup – SPRIselect (page 87)

30 min6.4 Post-Library Construction QC (page 88)

50 minTab 5 Text

Step 6: Visium HD Spatial Gene Expression – Probe-based Library Construction (page 81)-20°C long-term

Tab 6 Text

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Stepwise Objectives

Tab 2 Text
Tab 3 Text

Human or mouse whole transcriptome probe panels, consisting of ~3 pairs ofspecific probes for each targeted gene are added to the tissue, enablinghybridization and ligation of each probe pair. Tissue slides and Visium HD
Slides are loaded into the Visium CytAssist instrument, where they arebrought into proximity with one another. Gene expression probes are released
from the tissue upon CytAssist-Enabled Probe Release & Capture, enabling
capture by the spatially-barcoded oligonucleotides present in a hydrogel on
the Visium slide surface. The Visium HD Slide is removed from the Visium
CytAssist for downstream library preparation. Gene expression libraries are
generated from each tissue section and sequenced. Spatial Barcodes are used
to associate the reads back to the tissue section images for spatial mapping ofgene expression.

Tab 1 Text

The Visium HD Spatial Gene Expression assay is designed to analyze mRNAin tissue sections derived from formalin fixed & paraffin embedded (FFPE),
fresh frozen (FF), or fixed frozen (FxF) tissue samples. It uses probes targeting
the whole transcriptome. Each Visium HD Slide contains Capture Areas withbarcoded squares that include oligonucleotides required to capture gene
expression probes. Before the assay, tissue sections are processed as describedin their relevant Demonstrated Protocol. See Workflow Overview for
documentation references.

Tab 4 Text
Tab 5 Text
Tab 6 Text

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Visium HD Slides

is a continuous lawn of oligos comprised of 2 µm barcoded squares. Eachbarcoded square has oligos with the following composition:lTab 2 Text

Illumina TruSeq partial read 1 sequencing primer, unique molecular
identifier (UMI), Spatial Barcode, 30 nt poly(dT) sequence (captures
ligation product).

Tab 1 Text

The Visium HD Slide, 6.5 mm has 2 Capture Areas. Each Capture Area is 6.5 x
6.5 mm and is surrounded by a fiducial frame that is used for imagealignment. The fiducial frame + Capture Area is 8 x 8 mm. The Capture Area

Each Capture Area is surrounded by a spacer. This spacer creates a reaction
chamber that facilitates proper reagent addition.
The active surface of the slide is defined by an etched label that includes theserial number. The label should be legible. If the label appears reversed, theactive surface is facing down. The slide should be loaded onto the instrumentwith the active surface facing up.

Tab 3 Text
Tab 4 Text
Tab 5 Text
Tab 6 Text

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Step 1: Probe Hybridization
Tab 1 Text

The human or mouse whole transcriptome probe panel, consisting of ~3
specific probes for each targeted gene, is added to the deparaffinized,
destained, and decrosslinked tissues. Together, probe pairs hybridize to theircomplimentary target RNA.

Tab 2 Text
Tab 3 Text
Tab 4 Text
Tab 5 Text
Tab 6 Text

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Step 2: Probe Ligation
Tab 1 Text

After hybridization, a ligase is added to bridge the junction between the probe
pairs that have hybridized to RNA, forming a ligation product.

Tab 2 Text

Visium HD Slides are thawed, washed, and equilibrated prior to placement on
the Visium CytAssist instrument.

Tab 3 Text

Step 3: Visium HD Slide Preparation

Tab 4 Text
Tab 5 Text
Tab 6 Text

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Step 4: Probe Release & Extension
Tab 1 Text

Probe release and capture occurs in the Visium CytAssist instrument. The
single stranded ligation products are released from the tissue upon RNase
treatment and captured on the Visium slide. Once ligation products arecaptured, the slides can be removed from the instrument.
Ligation products are extended by the addition of the Spatial Barcode, UMI,
and partial Read 1 primer. This generates spatially-barcoded ligation products,
which can then be carried forward for library preparation.

Tab 2 Text
Tab 3 Text
Tab 4 Text
Tab 5 Text
Tab 6 Text

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Step 5: Pre-amplification and SPRIselectTab 1 Text

To generate ample material for library construction, barcoded ligation
products are amplified. This pre-amplification is followed by SPRIselectcleanup.

Tab 2 Text
Tab 3 Text

Pre-amplification material is collected for qPCR to determine sample index
PCR cycle number for gene expression libraries. The amplified material thenundergoes indexing via sample index PCR to generate final library molecules.
The final libraries are cleaned up by SPRIselect, assessed on a Bioanalyzer ora similar instrument, quantified by qPCR, and then sequenced.

Tab 4 Text

Step 6: Visium HD Spatial Gene Expression - Probebased Library Construction

Tab 5 Text
Tab 6 Text

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Sequencing
Tab 1 Text

A Visium HD Spatial Gene Expression - Probe-based library comprises
standard Illumina paired-end constructs, which begin and end with P5 and P7
adaptors. The 43 bp UMI and Spatial Barcode are encoded in TruSeq Read 1,
while Small RNA Read 2 (Read 2S) is used to sequence the ligated probe
product.
Illumina sequencer compatibility, sample indices, and library loading
information are summarized in the Sequencing section.

Tab 2 Text
Tab 3 Text

See Oligonucleotide Sequences on page 122

Tab 4 Text
Tab 5 Text
Tab 6 Text

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Tips & Best Practices
General Reagent Handling

23

Visium Slide Storage

23

Pipette Calibration

23

Visium HD Slide Handling23

Tissue Slide Handling

24

Reagent Addition to & Removal from Wells

25

Visium Slide Seal Application & Removal

27

Slide Incubation Guidance

28

Cassette Incubation

29

10x Magnetic Separator

30

Magnetic Bead Cleanup Steps

30

SPRIselect Cleanup & Size Selection

31

Sample Indices (i5/i7) in Sample Index PCR

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Icons

Signifies criticalstep requiring
accurate
execution

Troubleshooting
section includes
additional
guidance

Tab 1 Text

Tips & Best
Practices section
includes additional
guidance

Indicates a versionspecific update in volume,
temperature, instruction,etc.

Tab 2 Text

General Reagent Handlingll

Fully thaw and thoroughly mix reagents before use.
When pipette mixing reagents, unless otherwise specified, set pipette to
75% of total volume.

Tab 3 Text

Visium Slide Storagel

Do not open the mylar bag containing the Visium Spatial slides until
slides are ready to be used.

Pipette Calibration
Follow manufacturer’s calibration and maintenance schedules.

l

Pipette accuracy is particularly important when using SPRIselect reagents.

Tab 4 TextlVisium HD Slide Handlinglll

Wipe Visium HD Slide Mailer with a lint-free laboratory wipe to ease inhandling.
Prior to loading the Visium HD Slide on the CytAssist instrument, Visium
HD Slides will be thawed, washed, equilibrated, and dried. Using an
external light source (like a flashlight) can help confirm that the
Visium HD Slide is fully dried.

Tab 6 Text

Occasionally, small chips can arise on the edge of the Visium HD Slide.
These are mainly cosmetic in nature and do not typically impact assay
performance.
Sharp edges on the slide's corners may cause injury or damage if

Tips & Best Practices

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Tab 5 TextlKeep Visium HD Slide at –80°C until ready to use.

23

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mishandled. Handle with care. Wear proper PPE when handling and avoid
contact with the chipped corner to prevent cuts or scratches.

Tab 1 Text
Tab 2 Text

l

l

l

Visium HD Slides should only be washed if proceeding with a CytAssist
instrument run.
Always wear gloves when handling slides.
Ensure that the active surface of a slide faces up and is never touched.
The active surface is defined by a readable label. The image of thechipped Visium HD slide demonstrates the active surface facing up.
Minimize exposure of the slides to sources of particles and fibers.

lWhen pipetting reagent onto a slide, avoid generating bubbles.

l

If debris appears on the Visium HD Slide after performing slide washes,slide may be re-immersed in 0.1X SSC in the slider mailer to remove
debris before proceeding. If performing an additional wash, ensure theslide is dried as described in the protocol.
If necessary, unwashed, room temperature Visium HD Slides may bereturned to –80°C. Protect from light. Do not exceed 3 h at room
temperature. Do not exceed three freeze/thaw cycles.

Tab 5 TextlTab 4 Textll

Tab 3 TextlVisium HD Slide preparation steps should be done gently to avoid
damaging the slide.

Do not allow the Visium HD Slide to dry, except where indicated in the
protocol steps.

Tissue Slide HandlinglTips & Best Practices

To ensure compatibility with the Visium CytAssist, tissue sections must beplaced in specific areas on a blank slide. Tested slides, as well as
appropriate tissue placement areas, are listed in the Visium HD Protocol
Planner (CG000698).
Always wear gloves when handling slides.

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Tab 6 Textl

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Reagent Addition to & Removal from Wells
l

l

l

l

DO NOT forcefully insert pipette tip into well corners to avoid damaging
the Visium HD slide.
If applicable, perform washes next to the thermal cycler to avoid
significant changes in temperature during washes.
When processing two or more Visium Tissue Slide Cassettes, remove andadd reagent from the first cassette before proceeding to the next cassette.
Ensure tissue sections are always covered with reagent in between
removal and addition steps to avoid drying out of tissue samples.
Always cover the Capture Area or tissue completely when adding reagentsto the well. A gentle tap may help spread the reagent more evenly.

Tab 3 TextlDispense and remove reagents along the side of the wells without
touching the slide surface, tissue sections (when applicable), and without
introducing bubbles.

Tab 2 TextlAssemble slide into the cassette flat on a clean work surface.

Tab 1 Text

Reagent Addition

Tab 4 Text
Tab 5 Text
Tab 6 Text

Tips & Best Practices

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Reagent Removal from Wells
Slightly tilt the cassette while removing the reagent.

lPlace the pipette tip on the bottom edge of the wells.

ll

Tab 1 TextlRemove reagents along the side of the wells without touching the tissue
sections (when applicable) and without introducing bubbles.

Tab 2 Text

Remove all liquid from the wells in each step. To ensure complete
removal, check the bottom of the well by tilting the cassette slightly. A
meniscus at the bottom of the well will indicate the presence of liquid in
the well. Repeat removal steps until no reagent remains.

Tab 3 Text
Tab 4 Text
Tab 5 Text
Tab 6 Text

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Visium Slide Seal Application & Removall

If applying a Visium Slide Seal to a Tissue Slide Cassette, the seal must be
cut in half lengthwise. Cut the seal as shown in the image below. Six precut seals per tissue slide are necessary for this assay.

Tab 1 Text

Application

Tab 2 Text

l

l

l

Remove the back of the adhesive Visium Slide Seal.
Align the Visium Slide Seal with the surface of the cassette and apply
while firmly holding the cassette with one hand.
Press on the Visium Slide Seal to ensure uniform adhesion.
Use a fresh Visium Slide Seal when prompted during the protocol. Stepsthat do not require a new slide seal will specify that the slide seal shouldbe pulled back and reapplied instead.

Tab 4 TextlPlace the cassette flat on a clean work surface. Ensure surface of cassetteis dry.

Tab 3 TextlTab 5 Text
Tab 6 Text

Tips & Best Practices

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RemovallGrasp a Visium Slide Seal corner. Carefully pull on the Visium Slide Seal
up and across the cassette while firmly holding the cassette. Ensure thatno liquid splashes out of the wells. Movement of the gasket during slide
seal removal could damage the tissue section.

Tab 1 TextlPlace the cassette flat on a clean work surface.

Tab 2 Text
Tab 3 Text

Slide Incubation GuidancellPosition a Low Profile Thermocycler Adapter on a thermal cycler that is
set at the incubation temperature.
Ensure that the Low Profile Thermocycler Adapter is in contact with thethermal cycler block surface uniformly. Pre-equilibrate adapters at the
HOLD temperature described in the protocol steps for at least fiveminutes.
Open and close the thermal cycler lid gently. If the lid is adjustable,
tighten lid only as much as necessary. Avoid overtightening. Image below
is for demonstration purposes - thermal cycler lid should be closed whenincubating the cassette.

Tab 5 TextlTab 4 Text

Incubation at a Specified Temperature

Incubation at Room Temperaturel

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Tab 6 Text

Tips & Best Practices

Place the slide/cassette on a flat, clean, nonabsorbent work surface.

28

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Cassette Incubationlll

Ensure that the Low Profile Thermocycler Adapter is in contact with thethermal cycler surface uniformly. Consult the Visium HD Spatial Gene
Expression Protocol Planner (CG000698) for information on thermal cycler
compatibility.
Ensure that the cassette sits flush with the raised portion of the Low
Profile Thermocycler Adapter.

Tab 2 TextlPosition a Low Profile Thermocycler Adapter on a thermal cycler that is
set at the incubation temperature. Low Profile Thermocycler Adapter mustcome to temperature before cassette placement.

Tab 1 Text

Incubation using a Thermal Cycler:

Allow Low Profile Thermocycler Adapter to cool before removing fromthermal cycler.

Tab 3 Text
Tab 4 Text

Tissue Detachment on Tissue SlidesllEnsure that tissue sections are on Superfrost Plus or positively charged
slides. A list of tested slides can be found in the Visium HD Protocol
Planner (CG000698).
10x Genomics recommends placing tissues with large amounts ofconnective tissue (like breast or colon) on Schott Nexterion Slide H - 3D
Hydrogel Coated Slides (Schott North America, PN-1800434) to minimize
risk of tissue detachment. Schott H slides may also be used if priordetachment has been observed with tissues of interest.

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Tab 6 Text

Tips & Best Practices

Monitor section adhesion on tissue slides throughout the workflow.

Tab 5 Textl

User Guide | Visium HD Spatial Gene Expression Reagent KitsolFollow manufacturer instructions for Schott Nexterion Slide H slides,
which have specific requirements for storage and equilibration.

Tissue detachment before the completion of Probe Release during theworkflow can negatively impact performance. Ensure that all guidance intissue preparation handbook is followed.
For more information, see Troubleshooting on page 101.

Tab 1 TextlCG000685 | Rev B

10x Magnetic SeparatorllImages below are illustrative - actual appearance of magnetic separator
may vary. Guidance applies to all 10x Magnetic Separators.
Offers two positions of the magnets (high and low) relative to a tube,
depending on its orientation.
Flip the magnetic separator over to switch between high (magnet•High) or
low (magnet•Low) positions.

Tab 3 TextlTab 2 Textl

If using MicroAmp 8-Tube Strips, use the high position (magnet•High)
only throughout the protocol.

Tab 4 Text
Tab 5 Text

Magnetic Bead Cleanup StepslTips & Best Practices

The time needed for the solution to clear may vary based on specific step,
reagents, volume of reagents, etc.

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Tab 6 TextlDuring magnetic bead-based cleanup steps that specify waiting “until thesolution clears”, visually confirm clearing of solution before proceeding to
the next step. See panel below for an example. If solution is not clear,
leave on magnet until separation is complete.

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Images below are illustrative - actual appearance of magnetic separator
may vary. Guidance applies to all 10x Magnetic Separators.

Tab 1 Text
Tab 2 TextllPipette mix thoroughly as insufficient mixing of sample and SPRIselectreagent will lead to inconsistent results.
Use fresh preparations of 80% Ethanol.

Tab 4 TextlAfter aspirating the desired volume of SPRIselect reagent, examine thepipette tips before dispensing to ensure the correct volume is transferred.

Sample Indices (i5/i7) in Sample Index PCRll

Tab 3 Text

SPRIselect Cleanup & Size Selection

Choose the appropriate sample index sets to ensure that no sample indicesoverlap in a multiplexed sequencing run.

l

To avoid the risk of cross-contamination, use each plate well only once.

l

Record the index plate well location for each sample.

Tab 5 Text

Each well in the Dual Index Plate TS Set A contains a unique i7 and aunique i5 oligonucleotide.

Tab 6 Text

Tips & Best Practices

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Visium CytAssist
Instrument Orientation

33

Instrument Loading Guidelines

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Instrument Orientation
Tab 1 Text

Firmware version 2.0.0 or higher is required in the Visium CytAssist used forthis protocol.

Tab 2 Text
Tab 3 Text
Tab 4 Text
Tab 5 Text
Tab 6 Text

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Tab 1 Text
Tab 2 Text

Instrument Loading GuidelinesllEnsure tissue section placement is compatible with Visium CytAssist byusing the Visium CytAssist Accessory Kit Quick Reference Card
(CG000548). A list of tested slide types can be found in the Visium
HD Spatial Gene Expression Protocol Planner (CG000698).

Tab 3 TextlConsult the Visium CytAssist Training Kit UG (CG000549) for information
on practicing tissue slide loading.
Each tissue slide may be used for one Capture Area on a Visium HD Slide.

Tab 4 Text

Tissue Slide Loadinga. Ensure the tissue on each slide fits within the alignment guides of the
Tissue Slide Stage.
b. Align tissue within the center of the 6.5 mm slide alignment guides(rectangles) on either the left or right side of the stage.

Tab 5 Text
Tab 6 Text

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Tab 1 Text
Tab 2 Text
Tab 3 Text
Tab 4 Text

Alignment guide dimensions and locations are designed with a hightolerance to minimize data loss. Tissue within the alignment guides is

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Tab 6 Text

When placing a tissue slide on the Tissue Slide Stage, center the tissue orarea of interest within the area inside the outer edges of the alignment
guides (area in green in the images).

Tab 5 Text

The outside of the alignment guides, shown in red, mark the innerboundaries of the fiducial frame on a Capture Area. They mark a distanceapproximately 6 mm (6.5 mm slides) or 10.5 mm (11 mm slides) wide oneach side and surround the target region ensured to align within thefiducial frame. Though the Visium CytAssist instrument displays guides
for 11 mm Capture Areas, the Visium HD Spatial Gene Expression Assay is
only compatible with 6.5 mm Capture Areas.

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Tab 1 Text

ensured to overlap with the Capture Area during a successful run. Tissue
outside of these guides may be captured (area in yellow in the images),but it is not ensured. 10x Genomics recommends placing high-priority
tissue areas within the alignment guides.

Tab 2 Text
Tab 3 Text
Tab 4 Text
Tab 5 Text
Tab 6 Text

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Tab 1 Text
Tab 2 Text

c. If necessary, rotate the slide 180° as shown. Place off-center tissues closer
to the center line (shown as a blue line in the image below). Slides should
not overlap the center line.

Tab 3 Text
Tab 4 Text
Tab 5 Text

The image below demonstrates how movement of the Tissue Slides affects
where target molecules will end up on the Visium slide.

Tab 6 Text

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Tab 1 Text
Tab 2 Text

d. Press down on the wide end of clip to lift and pivot the narrow end of theclip. Pivot the clips to their outermost position.

Tab 3 Text
Tab 4 Text

f. Use fingers to finely adjust the position of the tissue within the alignment
guides.

Tab 5 Text

e. Lay the tissue slide flat against the stage surface. Both hands needed: use
one hand to hold the slide in place and the other to pivot the clips andoverlap the slide. Ensure that at least one clip secures the slide before
adjusting the position further.

Tab 6 Text

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Tab 1 Text
Tab 2 Text
Tab 3 Text

g. If only one tissue on one slide will be analyzed, use a blank slide for thesecond position on the Tissue Slide Stage. If using only one tissue slide,the unused Capture Area on the Visium HD Slide cannot be used inanother instrument run.

Tab 4 Text
Tab 5 Text
Tab 6 Text

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Visium HD Slide Loading

a. Open the Visium Slide Lock by using one finger to pivot.

Tab 1 Text

Before loading, record the slide serial number and note which tissue is placedin which Capture Area. Note which Capture Area will be placed on which sideof the Visium Slide Stage (Left = D1, Right = A1).

b. Line up the slide with the label face up and oriented to the right.
c. Fit the slide within the raised grooves on the left, top, and bottom.

Tab 2 Text
Tab 3 Text
Tab 4 Text

d. Hold slide in place with one hand while slowly closing the Visium Slide
Lock. Lock will partially obscure the slide label when correctly secured.
Close lock gently to avoid slide damage. In the images below, the slide(red outline) does not line up with the stage (blue outline).

Tab 5 Text
Tab 6 Text

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Tab 1 Text
Tab 2 Text
Tab 3 Text
Tab 4 Text
Tab 5 Text
Tab 6 Text

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Sample Preparation &Staining Guidelines
Sample Preparation

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Sample Preparation
Tab 1 Text

Proper tissue handling and preparation techniques preserve themorphological quality of the tissue sections and the integrity of mRNAtranscripts. Maintaining high-quality RNA is critical to assay performance.
Before performing the protocol steps in this User Guide, ensure that all
samples have followed the appropriate Tissue Preparation protocols as
described in the Workflow Overview. Listed below are key considerations
described in these tissue preparation protocols.

Tab 2 Text

Key Considerations
FFPE Tissue Sectioning & Section Placement
Assess RNA quality of the FFPE tissue block or from archived sections.
Practice sectioning and section placement with a nonexperimental block before placing
sections on slides.
Section the tissue block and place sections on compatible blank slides using a water bath.

Tissue Slide HandlingTab 3 Text

Place tissue sections on the allowable area on the slide. Allowable area may vary depending
on slide choice.

Ensure slides are completely dried after tissue placement, using a fan if necessary.
Store the slides containing FFPE sections for up to six months in a desiccator at room
temperature.

Tissue Slide Processing
After preparing tissue slides, process sections according to the desired staining protocols inthe Visium HD FFPE Tissue Preparation Handbook (CG000684). The handbook contains
information on tissue slide staining and imaging.

Tab 4 Text

After preparing tissue slides, stain sections according to the desired staining protocol in the
Visium HD FFPE Tissue Preparation Handbook (CG000684).

Key Considerations for FF Samples

Tab 5 Text

Freezing and Embedding
Snap freeze samples in a bath of isopentane and liquid nitrogen.
Embed frozen samples in OCT.
Store frozen samples in a sealed container at -80°C for long-term storage.
Slide Handling
Equilibrate slides to cryostat temperature before cryosectioning.

Tab 6 Text

Cryosectioning
Assess RNA quality of the tissue block.
Optional - assess tissue morphology via DAPI and H&E staining.

Sample Preparation & Staining Guidelines

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Key Considerations for FF Samples

Tab 1 Text

Practice sectioning and section placement with a nonexperimental block before placing
sections on slides.
Equilibrate OCT tissue block to the cryostat chamber temperature for 30 min.
Section the tissue block and place sections on compatible blank slides.
Tissue Slide Processing
After preparing tissue slides, process sections according to the desired staining protocols inthe Visium HD FF Tissue Preparation Handbook (CG000763). The handbook contains
information on tissue slide staining and imaging.

Tab 2 Text

Tissue Slide Handling
Maintain slides containing sections in a low moisture environment.
Keep slides cold and transport slides on dry ice.
Store tissue slides in a slide mailer at -80°C for up to two months.

Key Considerations for FxF Samples

Tab 3 Text

Fixation, Embedding, and Freezing
Fix samples in 4% PFA or formaldehyde
Cryopreserve samples in a sucrose gradient.
Embed fixed samples in OCT and freeze.
Sample Block Storage
Store frozen samples in a sealed container at -80°C for long-term storage.

Tab 4 Text

Slide Handling
Equilibrate slides to cryostat temperature before cryosectioning.
Cryosectioning
Assess RNA quality of the tissue block.
Optional - assess tissue morphology via DAPI and H&E staining.

Tab 5 Text

Practice sectioning and section placement with a nonexperimental block before placing
sections on slides.
Equilibrate OCT tissue block to the cryostat chamber temperature for 30 min.
Section the tissue block and place sections on compatible blank slides.
Tissue Slide Processing
After preparing tissue slides, process sections according to the desired staining protocols inthe Visium HD FxF Tissue Preparation Handbook (CG000764). The handbook contains
information on tissue slide staining and imaging.

Tab 6 Text

Tissue Slide Handling
Maintain slides containing sections in a low moisture environment.
Keep slides cold and transport slides on dry ice.
Store tissue slides in a slide mailer at -80°C for up to two months.

Sample Preparation & Staining Guidelines

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Step 1:
Probe Hybridization
1.0 Get Started

46

1.1 Probe Hybridization

47

1

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1.0 Get Started

Items

10x PN

Preparation & HandlingTab 1 Text

Each 10x Genomics reagent tube is enough for two 6.5 mm Visium HD Slides(four Tissue Slides). Number of reactions in reagent preparation tables refers
to the number of Tissue Slides.
Storage

Equilibrate to room temperature
Tube: 2000423
Kit: 1000466 or
1000667

Thaw at room temperature. If
precipitate persists, heat at 37°C until
dissolved. Avoid vortexing to prevent
bubble formation. Pipette mix 10x.
Keep the buffer at room temperature
after thawing and while performing theworkflow.

−20°C

Human
WT Probes
v2 - RHS

Tube: 2000657
Kit: 1000466

Thaw at room temperature. Vortex andcentrifuge briefly. Maintain at room
temperature.

−20°C

Human
WT Probes
v2 - LHS

Tube: 2000658
Kit: 1000466

Thaw at room temperature. Vortex andcentrifuge briefly. Maintain at room
temperature.

−20°C

Mouse
WT Probes
v2 - RHS

Tube: 2000913
Kit: 1000667

Thaw at room temperature. Vortex andcentrifuge briefly. Maintain at room
temperature.

−20°C

Mouse
WT Probes
v2 - LHS

Tube: 2000912
Kit: 1000667

Thaw at room temperature. Vortex andcentrifuge briefly. Maintain at room
temperature.

−20°C

Nucleasefree Water--

Ambient

10X PBS, pH
7.4

-

-

Ambient

Visium Slide
Seals

Component:
2000284
Kit: 1000669

See Tips & Best Practices. 3 pre-cut
slide seals are necessary for thecomplete workflow.

Ambient

10% Tween20--

Ambient

Tab 2 Text

FFPE Hyb
BufferTab 3 Text
Tab 4 Text

ObtainTab 5 Text
Tab 6 Text

Step 1: Probe Hybridization

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1.1 Probe Hybridizationa. Prepare Pre-Hybridization Mix according to the table shortly before use.
Add reagents in the order listed. Vortex and centrifuge briefly. Maintain atroom temperature.
10x PN1X(µl)

2X +10%(µl)

4X +10%(µl)

Nuclease-free Water

-

134.2

295.3

590.6

10X PBS, pH 7.4

-

15.0

33.0

66.0

10% Tween-20

-

0.8

1.7

3.4

Total

-

150.0

330.0

660.0

c. Peel back Visium Slide Seals.
d. Using a pipette, remove all buffer from each well at well corners. Use a
P20 pipette to remove any remaining liquid. See image below for properliquid removal technique.

Tab 3 Text

b. Retrieve Tissue Slide Cassettes containing H&E stained or IF stained
sections.

Tab 2 Text

Pre-Hybridization Mix

Tab 1 Text

During reagent removal steps, ensure that ALL liquid is removed from wells.
Use a P20 pipette to remove any remaining liquid. See Tips & Best Practices
for guidance on Reagent Removal.

Tab 4 Text
Tab 5 Text

f. Re-apply Visium Slide Seal on each Tissue Slide Cassette.

Step 1: Probe Hybridization

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Tab 6 Text

e. Add 150 µl Pre-Hybridization Mix along the side of each well to uniformly
cover tissue sections, without introducing bubbles.

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g. Incubate for 15 min at room temperature.

Tab 1 Text

Reaction Volume

Run Time

50°C

100 μlOvernight (16 - 24 h)

Step

Temperature

Time hh:mm:ss

Pre-equilibrate

50°C

Hold

Hybridization

50°C

Overnight (16 - 24 h)

Post Hybridization Wash

50°C

Hold

Tab 3 Text

Lid Temperature

2X +10%(µl)

4X +10%
(µl)

-

10.0

22.0

44.0

FFPE Hyb Buffer

2000423

70.0

154.0

308.0

Human WT Probes v2 - RHS
orMouse WT Probes v2 - RHS

2000657or
2000913

10.0

22.0

44.0

Human WT Probes v2 - LHS
orMouse WT Probes v2 - LHS

2000658or
2000912

10.0

22.0

44.0

-

100.0

220.0

440.0

Nuclease-free Water

Total

j. Remove all Pre-Hybridization Mix from each well.
k. Add 100 µl room temperature Probe Hybridization Mix to each well.

Step 1: Probe Hybridization

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Tab 6 Text

1X
(µl)

Tab 5 Text

10x PN

Tab 4 Text

i. Prepare Probe Hybridization Mix according to the table shortly before use.
Add reagents in the order listed. Keep at room temperature. Pipette mix
10x and centrifuge briefly.
Probe Hybridization Mix

Tab 2 Text

h. Place a Low Profile Thermocycler Adapter in the thermal cycler. Prepare
thermal cycler with the following incubation protocol and start the
program. See Tips & Best Practices for more information on cassette
incubation in a thermal cycler.

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Tab 1 Text

l. Apply a new pre-cut Visium Slide Seal to each Tissue Slide Cassette andplace on the Low Profile Thermocycler Adapter on the thermal cycler.
Close the thermal cycler lid.

Tab 2 Text
Tab 3 Text

m. Skip Pre-equilibrate step to initiate Hybridization.

Tab 4 Text
Tab 5 Text
Tab 6 Text

Step 1: Probe Hybridization

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Step 2:
Probe Ligation
2.0 Get Started

51

2.1 Post-Hybridization Wash

52

2.2 Probe Ligation

54

2.3 Post-Ligation Wash

55

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2.0 Get Started

Items

10x PN

Preparation & HandlingTab 1 Text

Each 10x Genomics reagent tube is enough for two 6.5 mm Visium HD Slides(four Tissue Slides). Number of reactions in reagent preparation tables refers
to the number of Tissue Slides.
Storage

Equilibrate to room temperature
Tube: 2000424
Kit: 1000466 or
1000667

Thaw at room temperature. If
precipitate persists, heat at 37°C until
dissolved. Vortex briefly. Flick tube
until liquid settles at the bottom of the
tube.

−20°C

2X Probe
Ligation
BufferTube: 2000445
Kit: 1000466 or
1000667

Thaw at room temperature until no
precipitate remains. Vortex andcentrifuge briefly.

−20°C

Post-Ligation
Wash Buffer

Tube: 2000419
Kit: 1000466 or
1000667

Thaw at room temperature. If
precipitate persists, heat at 37°C until
dissolved. Vortex.

−20°C

Probe
LigationEnzymeTube: 2000425
Kit: 1000466 or
1000667

Centrifuge briefly. Maintain on ice.

−20°C

Nucleasefree Water--

Ambient

20X SSC
Buffer

--Ambient

Tab 2 Text

FFPE PostHyb Wash
BufferTab 3 Text

Place on ice

ObtainTab 4 Text
Tab 5 Text
Tab 6 Text

Step 2: Probe Ligation

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2.1 Post-Hybridization Wash

b. Prepare 2X SSC Buffer according to the table. Add reagents in the order
listed. Maintain at room temperature. DO NOT discard excess buffer, as
it will be used for subsequent washes in the protocol.
2X +10%(µl)

4X +10%
(µl)

Stock

Final

SSC

20X

2X

150

315

615

Nuclease-free Water

-

-

1,350

2,835

5,535

Total

-

1,500*

3,150*

6,150*Tab 2 Text

1X
(µl)

SSC Buffer

Tab 1 Text

a. Aliquot FFPE Post-Hyb Wash Buffer (495 μl/per 6.5 mm sample) andpre-heat to 50°C in a water bath or thermomixer. Maintain pre-heatedwash buffer at 50°C throughout washes.

c. Remove Visium Tissue Slide Cassettes from Low Profile Thermocycler
Adapter and place on a flat, clean work surface.

Tab 3 Text

d. Peel back Visium Slide Seals and using a pipette, remove all Probe
Hybridization Mix from each well. See Tips and Best Practices for slide
seal removal guidance.

Tab 4 Text
Tab 5 Text

e. Immediately add 150 µl pre-heated FFPE Post-Hyb Wash Buffer to eachwell. Avoid well drying or cooling to room temperature. Removal andaddition of buffers should be done quickly.

Step 2: Probe Ligation

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Tab 6 Text

f. Re-apply Visium Slide Seal on each Tissue Slide Cassette and place on Low
Profile Thermocycler Adapter on pre-heated thermal cycler. Close thermal

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cycler lid. See Tips & Best Practices for more information on cassette
incubation on a thermal cycler.

Tab 1 Text

Ensure that Tissue Slide Cassette sits snugly on the Low Profile
Thermocycler Adapter for all wash steps.
g. Skip the Hybridization step on thermal cycler and initiate PostHybridization Wash.
h. Incubate Visium Tissue Slide Cassettes in thermal cycler at 50°C for 5
min.

Tab 2 Text

i. Remove Tissue Slide Cassettes from Low Profile Thermocycler Adapter
and place on a flat, clean work surface. Exercise caution, as slide is hot.
j. Peel back Visium Slide Seals and using a pipette, remove all FFPE PostHyb Wash Buffer from each well.
k. Immediately add 150 µl pre-heated FFPE Post-Hyb Wash Buffer to well.
Avoid well drying or cooling to room temperature. Removal and additionof buffers should be done quickly.

Tab 3 Text

l. Re-apply Visium Slide Seal on each Tissue Slide Cassette and place on Low
Profile Thermocycler Adapter on pre-heated thermal cycler. Close thermal
cycler lid.
m. Incubate in thermal cycler at 50°C for 5 min.
n. Repeat steps i-m one more time for a total of three washes.

p. Add 150 µl 2X SSC Buffer to each well and re-apply Visium Slide Seal.
q. Let the Tissue Slide Cassettes cool to room temperature (~3 min) before
proceeding to the next step.

Tab 4 Text

o. Peel back Visium Slide Seals and remove FFPE Post-Hyb Wash Buffer fromeach well.

Tab 5 Text
Tab 6 Text

Step 2: Probe Ligation

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2.2 Probe Ligation

Run Time

37°C (lid may be turned off if the instrument does not enable
37°C)

100 μl1h

Step

TemperatureTime
hh:mm:ss

Pre-equilibrate

37°C

Hold

Ligation

37°C

01:00:00

Hold

4°C

Hold

10x PN1X(µl)

2X +10%(µl)

4X +10%(µl)

Nuclease-free Water

-

24.0

52.8

105.6

2X Probe Ligation Buffer

2000445

30.0

66.0

132.0

Probe Ligation Enzyme

2000425

6.0

13.2

26.4

-

60.0

132.0

264.0

Total

c. Remove Visium Slide Seals from each Tissue Slide Cassette and remove all
2X SSC Buffer from each well.

e. Apply a new pre-cut Visium Slide Seal on each Tissue Slide Cassette andplace on the Low Profile Thermocycler Adapter on the pre-heated thermal
cycler. Close the thermal cycler lid.

Tab 5 Text

d. Add 60 µl Probe Ligation Mix directly to tissue sections in each well,without introducing bubbles. Tap Tissue Slide Cassettes gently to ensure
uniform coverage.

Tab 4 Text

Probe Ligation Mix

Tab 3 Text

b. Prepare Probe Ligation Mix according to the table shortly before use. Add
reagents in the order listed. Pipette mix 10x and centrifuge briefly.
DO NOT vortex. Maintain on ice.

Tab 2 Text

Reaction
VolumeLid Temperature

Tab 1 Text

a. Place a Low Profile Thermocycler Adapter onto a thermal cycler (if notalready placed), prepare with the following incubation protocol, and start
the program.

f. Skip Pre-equilibrate step to initiate Ligation.

Tab 6 Text

Step 2: Probe Ligation

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2.3 Post-Ligation Washa. If the stopping point after the Post-Ligation Wash will not be used, remove
Visium HD slide mailer from –80°C. Remove slide mailer from mylar bag.
Keep slide mailer upright, capped, and thaw at room temperature for
30–60 min. Two Visium HD slides may be prepared at the same time.

c. Remove the Tissue Slide Cassettes from the Low Profile Thermocycler
Adapter and place on a flat, clean work surface.

Tab 2 Text

b. Pre-heat Post-Ligation Wash Buffer (110 μl/sample) to 57°C. Only 100 μl
per 6.5 mm sample is needed.

Tab 1 Text

Use room temperature Post-Ligation Wash Buffer at first wash step (step 2.3f).
Use pre-heated Post-Ligation Wash Buffer at second wash step (step 2.3k).

d. Immediately prepare thermal cycler with the following incubation
protocol and start the program.
Reaction Volume

Run Time

57°C

100 μl-Step

Temperature

Time

Incubate

57°C

Hold

Tab 3 Text

Lid Temperature

e. Remove Visium Slide Seals from each Tissue Slide Cassette and using apipette, remove all Probe Ligation Mix from each well.

g. Apply a new pre-cut Visium Slide Seal on each Tissue Slide Cassette andplace on Low Profile Thermocycler Adapter on pre-heated thermal cycler.
Close thermal cycler lid. See Tips & Best Practices for more information on
cassette incubation in a thermal cycler.

h. Incubate at 57°C for 5 min.
i. Remove Tissue Slide Cassettes from the Low Profile Thermocycler Adapter
and place on a flat, clean work surface.

Tab 5 Text

Ensure that Tissue Slide Cassette sits snugly on the Low Profile
Thermocycler Adapter for all wash steps.

Tab 4 Text

f. Immediately add 100 µl room temperature Post-Ligation Wash Buffer toeach well. Removal and addition of buffers should be done quickly.

j. Peel back Visium Slide Seals and using a pipette, remove all Post-Ligation
Wash Buffer from each well.

Tab 6 Text

k. Add 100 µl pre-heated Post-Ligation Wash Buffer to each well.

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m. Incubate at 57°C for 5 min.
n. Remove Tissue Slide Cassettes from Low Profile Thermocycler Adapter
and place on a flat, clean work surface.

Tab 1 Text

l. Re-apply Visium Slide Seal on Tissue Slide Cassette and place on Low
Profile Thermocycler Adapter on pre-heated thermal cycler. Close thermal
cycler lid.

o. Peel back Visium Slide Seals and using a pipette, remove all Post Ligation
Wash Buffer from each well.

Tab 2 Text

p. Add 150 µl 2X SSC Buffer prepared in 2.1 Post-Hybridization Wash on
page 52 to each well.
q. Remove all 2X SSC Buffer from each well.
r. Add 150 µl 2X SSC Buffer to each well.
s. Re-apply Visium Slide Seals on each Tissue Slide Cassette.
t. Allow Tissue Slide Cassettes to come to room temperature for 5 min.

Tab 3 Text

u. Store at 4°C for up to 24 h or proceed to next step.

Tab 4 Text
Tab 5 Text
Tab 6 Text

Step 2: Probe Ligation

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Step 3:
Visium HD Slide Preparation
3.0 Get Started

58

3.1 Visium HD Slide Wash

59

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3.0 Get Started

Firmware version 2.0.0 or higher is required in the Visium CytAssist used forthis protocol.
Items

Preparation & Handling

Storage

Nucleasefree Water--

Ambient

20X SSC--

Ambient

VisiumCassette

Component: 3001830/
3001831
Kit: 1000669

See Cassette Assembly Quick
Reference Card (CG000577)

Ambient

ObtainTab 2 Text

10x PN

Tab 1 Text

Each 10x Genomics reagent tube is enough for two 6.5 mm Visium HD Slides.
Number of reactions in reagent preparation tables refers to the number of
Visium HD slides.

Tab 3 Text
Tab 4 Text
Tab 5 Text
Tab 6 Text

Step 3: Visium HD Slide Preparation

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3.1 Visium HD Slide Wash
Tab 1 Text

a. If Visium HD Slide has not been thawed, remove Visium HD slide mailer
from –80°C. Remove slide mailer from mylar bag. DO NOT uncap slide
mailer. Keep slide mailer upright and thaw at room temperature for 30–
60 min. Two Visium HD slides may be prepared at the same time.
DO NOT touch spacer during slide washes.
b. Prepare 0.1X SSC Buffer according to the table below. Pipette mix.
Maintain at room temperature.

Nuclease-free Water

Stock
-

Final
-

SSC

20X

Total

-

0.1X

1 Visium HD Slide(ml)

2 Visium HD Slides
(ml)

59.7

119.4

0.3

0.6

60.0

120.0

Tab 2 Text

0.1X SSC Buffer

c. Open slide mailer.

e. Add 7 ml 0.1X SSC Buffer slowly along the side of the mailer on the otherside of the slide, so that both sides of the slide are immersed in 0.1X SSC
Buffer.

Tab 3 Text

d. Add 7 ml 0.1X SSC Buffer slowly along the side of the mailer on one sideof the slide.

Tab 4 Text
Tab 5 Text

f. Incubate at room temperature for 1 min. DO NOT close the mailer.
g. Remove 0.1X SSC Buffer by securing slide in place with one finger andpouring SSC Buffer from mailer.

Tab 6 Text

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Tab 1 Text

j. Incubate at room temperature for 5 min.
k. Remove 0.1X SSC Buffer by securing slide in place with one finger andpouring SSC Buffer from mailer.

Tab 3 Text

i. Add 7 ml 0.1X SSC Buffer slowly along the side of the mailer on the otherside of the slide, so that both sides of the slide are immersed in 0.1X SSC
Buffer.

Tab 2 Text

h. Add 7 ml 0.1X SSC Buffer slowly along the side of the mailer on one sideof the slide.

l. Repeat h-k twice more for a total of three 5 min washes. During the lastwash, save SSC Buffer in the mailer in case Visium HD Slide needs
additional wash.

Tab 4 Text

m. Ensure back (side without spacers) of Visium HD Slide is dry. If necessary,remove excess moisture from the back of the slide with a lint-freelaboratory wipe.
n. Gently touch long edge of Visium HD slide on a lint-free laboratory wipe
3–5x to remove excess SSC buffer

Tab 5 Text
Tab 6 Text

o. Ensure Visium HD Slide is free of particulate matter. If necessary, wash

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Visium HD slide by immersing in slide mailer with 0.1X SSC buffer fromstep l.

p. Place Visium HD Slide in a new 6.5 mm Visium Cassette. See Visium
Cassette S3 Quick Reference Card (Document CG000730) for assembly
instructions.

Tab 1 Text

Record slide serial number before placing Visium HD slide into cassette.

q. Add 100 µl 0.1X SSC to each well in the cassette.
r. Apply a new pre-cut Visium Slide Seal on the Visium Cassette.

Tab 2 Text

s. Leave 0.1X SSC Buffer in the Visium Cassette at room temperature until
Visium HD Slide equilibration. DO NOT exceed 2 h before proceeding witha CytAssist run. Keep Visium Cassette and Visium HD slide free fromdebris.

Tab 3 Text
Tab 4 Text
Tab 5 Text
Tab 6 Text

Step 3: Visium HD Slide Preparation

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Step 4:
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4.0 Get Started

63

4.1 CytAssist-Enabled Probe Release & Capture

65

4.2 Probe Extension

75

4.3 Probe Elution

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4.0 Get Started
Tab 1 Text

Each 10x Genomics reagent tube is enough for two 6.5 mm Visium HD Slides(four tissue slides). Number of reactions in reagent preparation tables refers
to the number of Tissue Slides. After instrument run, number of reactions
refers to the number of wells in a Visium Cassette.
Ensure that the Visium CytAssist is powered on, running firmware version
2.0.0 or higher, is clean, and ready to perform an experimental run.
10x PN

Preparation & Handling

Storage

2X RNase Buffer

Tube: 2000411
Kit: 1000668

Thaw at room temperature.
Pipette mix slowly andthoroughly. DO NOT vortex.

−20°C

Extension Buffer

Tube: 2000409
Kit: 1000668

Thaw at room temperature,vortex, centrifuge briefly.

−20°C

Perm Enzyme B

Tube: 3000553
Kit: 1000668

Pipette mix, centrifuge briefly.
Maintain at room temperature
until ready to use. Perm Enzyme
B is added to the Probe Release
Mix immediately before running
the CytAssist instrument.

−20°C

RNase Enzyme

Tube: 3000605
Kit: 1000668

Pipette mix, centrifuge briefly.
Maintain on ice until ready to
use.

−20°C

Extension Enzyme

Tube: 2000389
Kit: 1000668

Pipette mix, centrifuge briefly.
Maintain on ice until ready to
use.

−20°C

Visium HD Slide-

Obtain washed Visium HD Slide.

Ambient

Nuclease-free
Water

-

-

Ambient

Tris 1 M, pH 8.0(Tris-HCl)-

Manufacturer's
recommendations.

Ambient

Alcoholic Eosin-

Manufacturer's
recommendations.

Ambient

10X PBS-Use 10X PBS stock to prepare
1X PBS.

Ambient

8 M KOH Solution-

Manufacturer's
recommendations.

Ambient

Qiagen Buffer EB-

Manufacturer's
recommendations.

Ambient

Visium Slide Seals

Component:
2000283
Kit: 1000669

See Tips & Best Practices.

Ambient

Equilibrate to room temperature

Tab 2 Text

Items

Tab 3 Text

Place on ice

63

Tab 6 Text

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Tab 5 Text

Step 4: Probe Release & Extension

Tab 4 Text

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Tab 1 Text
Tab 2 Text
Tab 3 Text
Tab 4 Text
Tab 5 Text
Tab 6 Text

Step 4: Probe Release & Extension

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4.1 CytAssist-Enabled Probe Release & Capture
a. Ensure that Visium CytAssist is powered on, clean, and ready for an
experimental run. Visium CytAssist firmware version 2.0.0 or higher isrequired for this protocol.

Tab 1 Text

If processing more than two tissue slides, keep remaining tissue slides at 4°C
with 2X SSC buffer. For more guidance, see Appendix on page 113.

The home screen is the most common state of the instrument. There areseveral key functions accessible directly from the home screen.

Tab 2 Text
Tab 3 Text

b. Press blue New Run button on the touchscreen to initiate run.
c. Enter the following assay configuration information:l

A slide scanner may be used to scan the Visium HD slide for
automatic serial number input. See Visium HD Spatial Gene
Expression Protocol Planner (CG000698) for compatible part numbers.
The slide scanner may be used to scan the bottom of the cassette if the
Visium HD slide is already assembled into the cassette.

Tab 5 TextlCustom run name, temperature, and time (37°C for 30 min isrecommended for most applications)d. Enter the following sample information:l

Tab 4 Text

Visium Slide serial number. Ensure serial number is accurate. If
serial number is entered in the wrong format, the check mark button
to proceed will be grayed out. Record serial number for future use.

Sample names and the locations of each sample on the instrument (A1for right side, D1 for left side)Tab 6 Text

Step 4: Probe Release & Extension

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Tab 1 Text

10x PN

2 Tissue Slides(µl) (includes overage)

4 Tissue Slides(µl) (includes overage)

Nuclease-free Water

-

5,940

11,880

10X PBS

-

660

1,320

Total

-

6,600

13,200

1X PBS

10% Eosin

10x PN1X(µl)

2X +10%(µl)

4X +10%
(µl)

-

15

33

66

1X PBS

-

135

297

594

Total

-

150

330

660

g. Prepare Slide Equilibration Mix according to the table below. Pipette mix
and centrifuge briefly. Maintain on ice.
Slide Equilibration Mix

10x PN

Nuclease-free Water

1X
(µl)

2X +10%(µl)

4X +10%
(µl)

44.0

96.8

193.6

2000411

50.0

110.0

220.0

RNase Enzyme

3000605

6.0

13.2

26.4

-

100.0

220.0

440.0

Total

Tab 5 Text

2X RNase Buffer

Tab 4 Text

Alcoholic Eosin

Tab 3 Text

f. Prepare 10% Eosin. Vortex and centrifuge briefly. Eosin should be
prepared fresh for each CytAssist instrument run.

Tab 2 Text

e. Prepare 1X PBS. Maintain at room temperature.

h. Retrieve Visium Cassette with Visium HD Slide.

Step 4: Probe Release & Extension

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Tab 6 Text

i. Remove Visium Slide Seal and using a P200 pipette, remove 0.1X SSC fromthe right corner of each well in the cassette without scratching the fiducial
frame or hydrogel. Use a P20 pipette to remove any remaining liquid. See
image below for proper liquid removal technique.

User Guide | Visium HD Spatial Gene Expression Reagent Kits

CG000685 | Rev B

Tab 1 Text
Tab 2 Text
Tab 3 Text

j. Add 100 µl Slide Equilibration Mix to each well in the cassette. Gently tap
to ensure uniform coverage of the Capture Area.
k. Apply a new uncut Visium Slide Seal on the Visium Cassette and incubate
at room temperature for 10 min.

Tab 4 Text

l. Prepare Probe Release Mix during Visium HD Slide equilibration.
DO NOT prepare Probe Release Mix for more than two tissue slides at atime. Pipette mix thoroughly until solution is homogenous. Avoid
generating bubbles. Maintain on ice.
10x PN

2 Tissue Slides(µl) (includes overage)

2X RNase Buffer

2000411

20.0

RNase Enzyme

3000605

17.5

-

37.5

Total

Tab 5 Text

Probe Release Mix

Tab 6 Text

m. Remove Visium Slide Seal and using a P200 pipette, remove Slide
Equilibration Mix from the right corner of each well in the cassette
without scratching the fiducial frame or hydrogel. Use a P20 pipette toremove any remaining liquid. See image in step i for proper liquid
removal technique.
Failure to remove Slide Equilibration Mix completely may result in
reduced assay performance.

Step 4: Probe Release & Extension

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Tab 1 Text

n. Remove top half of Visium Cassette, leaving Visium HD Slide resting inbottom half of Visium Cassette. Rest top half of Visium Cassette such thatthe gaskets face away from work surface, as shown in the image below.
See Visium Cassette S3 Quick Reference Card (CG000730) for more
information on cassette disassembly.

Tab 2 Text
Tab 3 Text

o. Remove Visium HD Slide from Visium Cassette. Avoid touching active
surface. If necessary, remove excess moisture from the back of the slidewith a lint-free laboratory wipe.
The time between completing step o and starting step q should notexceed 5 min.
Save Visium Cassette for use after the instrument run. Ensure back of
Visium HD Slide is dry.

Tab 4 Text

p. Load Visium HD Slide against the grooves of Visium Slide Stage, using one
hand to load the slide and the other to close the Visium Slide Lock.

Tab 5 Text
Tab 6 Text

Step 4: Probe Release & Extension

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Tab 1 Text
Tab 2 Text
Tab 4 Text

Ensure area around the Visium CytAssist instrument is free from debris.
While the Visium HD Slide is drying, prepare Tissue Slides as described insteps r-aa.

Tab 3 Text

q. Allow Visium HD Slide to dry on the Visium Slide Stage for 10 min.
Inspect entire well. If liquid remains on the slide or anywhere in the well,continue drying and proceed immediately when no liquid remains within
the well.

Tab 5 Text

r. Retrieve Tissue Slide Cassettes.

Tab 6 Text

s. Using a pipette, remove all 2X SSC Buffer from each well of the Tissue
Slide Cassettes.
t. Remove tissue slides from Tissue Slide Cassettes.
u. Add 100 µl 10% Eosin to uniformly cover each tissue section per slide.

Step 4: Probe Release & Extension

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v. Incubate 1 min at room temperature.

x. While holding the slide over the liquid waste container, rinse with 1 ml 1X
PBS. DO NOT pipette directly onto tissue.

Tab 1 Text

w. Remove 10% Eosin by holding slide at an angle over a liquid waste
container.

Tab 2 Text

z. Gently waft/flick slide back and forth to remove excess PBS. Remove any
excess PBS with a lint-free laboratory wipe in areas outside of the tissue,
without touching the tissue sections.

Tab 3 Text

y. Repeat step x two more times for a total of three washes.

Tab 4 Text
Tab 5 Text

Complete tissue slide alignment before Visium HD slide is done drying.

Step 4: Probe Release & Extension

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Tab 6 Text

aa. Wipe back of tissue slides with a lint-free laboratory wipe and load into
Visium CytAssist. Ensure tissue section is completely dry prior to
instrument run. If necessary, repeat slide wafting. See Instrument Loading
Guidelines on page 34 for more information.

User Guide | Visium HD Spatial Gene Expression Reagent Kits

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Tab 1 Text
Tab 2 Text

Before proceeding to next step, ensure that Visium HD slide is completely
dry. See step q.

Tab 3 Text

ab. Pipette mix Perm Enzyme B (PN-3000553) and centrifuge briefly. Remove
Probe Release Mix from ice. Add 2.5 µl of Perm Enzyme B to 37.5 µl ofProbe Release Mix (prepared at step 4.1h). Pipette mix 15x with pipette set
to 30 µl. Avoid generating bubbles. Centrifuge for 5 sec.
The time between adding Perm Enzyme B to Probe Release Mix andstarting the Visium CytAssist instrument run should be less than 5 min.

ad. Slowly dispense 17 µl of Probe Release Mix into the center of each spacer
well on the Visium HD Slide, using a fresh pipette tip for each dispense.
Do not push plunger beyond first stop to avoid generating bubbles.

Tab 4 Text

ac. Slowly aspirate 17 µl of Probe Release Mix and inspect the pipette tip.
Ensure that no bubbles were drawn up into the pipette tip.

Tab 5 Text
Tab 6 Text

Step 4: Probe Release & Extension

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ae. Close lid and press Next.

Tab 1 Text

The home screen will now display a play symbol and run information
along the bottom of the screen.
af. Press play button to start the run. 37°C for 30 min is recommended formost applications.
lMidrun progress bar will show the time remaining in the run.

Tab 2 TextlTab 3 Text

Yellow before a run begins indicates a user-recoverable error. If anerror occurs, follow on-screen prompts.

ag. Place Low Profile Thermocycler Adapter in the thermal cycler. Prepare
thermal cycler with the following incubation protocol and start the
program.

53°C (lid may be turned off if the instrument does not enable53°C)

100 μl60 minStep

TemperatureTime

Pre-equilibrate

53°C

Hold

Probe Extension 1

53°C

00:30:00

Cool Down

4°C

00:03:00

Hold

4°C

Hold

Probe Extension 2

53°C

00:30:00

Cool Down

4°C

00:03:00

Hold

4°C

Hold

Tab 5 Text

Run Time

Tab 4 Text

Reaction
VolumeLid Temperature

ah. Prepare Probe Extension Mix. Pipette mix. Centrifuge briefly. Maintain on
ice.

Tab 6 Text

Step 4: Probe Release & Extension

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10x PN1X(µl)

2X +10%(µl)

4X +10%(µl)

Extension Buffer

2000409

147.0

323.4

646.8

Extension Enzyme

2000389

3.0

6.6

13.2

-

150.0

330.0

660.0

Total

Tab 1 Text

Probe Extension Mix

ai. At the end of a run, the button will display "Done" and a "Run Info" tab at
the bottom of the screen. DO NOT allow sample to sit in the Visium
CytAssist after run completion. Immediately move to next step.
Green indicates a successfully completed run.

l

Red indicates a failed run/errorlYellow at the end of a run indicates an incomplete run.

l

Tab 2 Textl

For more information on errors, consult the Visium CytAssist
Instrument User Guide (CG000542).

Tab 3 Text
Tab 4 Text
Tab 5 Text

aj. Click "Done" button and open lid. DO NOT power off the instrument at
this time, as it needs to process support data. It is normal for the Visium
slide to move after opening instrument.

Tab 6 Text

Step 4: Probe Release & Extension

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Tab 1 Text

al. While holding Visium HD Slide over liquid waste container, rinse each
Capture Area with 1 ml Buffer EB. DO NOT pipette directly onto Capture
Areas, which are surrounded by the fiducial frames.

Tab 3 Text

Rinse slide near Visium CytAssist instrument to ensure prompt washing of
Capture Areas.

Tab 2 Text

ak. Immediately remove Visium HD Slide. It is normal if tissue remains oftissue slides after run completion.

Tab 4 Text
Tab 5 Text

am. Repeat step al two more times for a total of three washes per Capture
Area.
an. Place Visium HD Slide in the same Visium Cassette from earlier in thisstep.
Some moisture remaining on the Visium HD Slide is normal.
ao. Proceed immediately to Probe Extension. CytAssist instrument should
only be cleaned and data transferred at a safe stopping point.

Tab 6 Text

Step 4: Probe Release & Extension

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4.2 Probe Extension
a. Add 75 µl Probe Extension Mix to each well. Gently tap Visium Cassette toensure uniform coverage of Capture Area. Keep remaining Probe
Extension Mix on ice.

Tab 1 Text

Probe Extension occurs over two rounds, Probe Extension 1 and Probe
Extension 2.

Tab 2 Text
Tab 3 Text

b. Apply new uncut Visium Slide Seal on Visium Cassette and place on the
Low Profile Thermocycler Adapter on pre-heated thermal cycler. Close
thermal cycler lid.
c. Skip Pre-equilibrate step to initiate Probe Extension 1.

Tab 4 Text

d. After the first Cool Down, open thermal cycler lid and remove cassettefrom Low Profile Thermocycler Adapter,e. Peel back Visium Slide Seal and remove Probe Extension Mix from eachwell.
f. Add 75 µl Probe Extension Mix to each well.
Gently tap Visium Cassette to ensure uniform coverage of Capture Area.

Tab 5 Text

g. Re-apply Visium Slide Seal and place cassette on Low Profile
Thermocycler Adapter. Close thermal cycler lid.
h. Skip Hold step to initiate Probe Extension 2.
i. Sample may remain at 4°C in the thermal cycler for up to 24 h.
j. Clean CytAssist instrument. Consult Visium CytAssist Instrument User
Guide (CG000542) for more information.

Tab 6 Text

Step 4: Probe Release & Extension

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4.3 Probe Elution
Tab 1 Text

a. Prepare 0.08 M KOH Mix according to the appropriate table shortly before
use, adding reagents in the order listed. Vortex and centrifuge briefly.
Maintain at room temperature.
6.5 mm Slides
Final

1X
(µl)

2X +10%(µl)

4X +10%(µl)

Nuclease-free Water

-

-

49.5

108.9

217.8

KOH

8M

0.08 M

0.5

1.1

2.2

Total

-

-

50.0

110.0

220.0

Tab 2 Text

Stock

KOH Mix

b. Remove Visium Cassette from Low Profile Thermocycler Adapter andplace on a flat, clean work surface after Probe Extension is complete.
c. Remove Visium Slide Seal and using a pipette, remove all Probe Extension
Mix from wells.

Tab 3 Text

d. Add 150 µl Buffer EB to each well.
e. Remove all Buffer EB from wells.
f. Add 50 μl 0.08 M KOH Mix to each well. Gently tap Visium Cassette toensure uniform coverage of Capture Area.
g. Incubate at room temperature for 10 min.

i. Transfer all solution for each sample containing the probes to a tube in an
8-tube strip containing 1 M Tris-HCl. Vortex, centrifuge briefly, and place
on ice. DO NOT leave behind any solution in the wells. See Tips & Best
Practices for reagent removal instructions.

Tab 4 Text

h. Add 3 μl 1 M Tris-HCl pH 8.0 to a tube in an 8-tube strip for each sample.

Tab 5 Text
Tab 6 Text

Step 4: Probe Release & Extension

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5.1 Pre-Amplification

79

5.2 Pre-Amplification Cleanup - SPRIselect80

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5.0 Get Started

Item

10x PN

Preparation & Handling

StorageTab 1 Text

Number of reactions in reagent preparation tables refers to the number ofsamples from Probe Elution.

Equilibrate to room temperature
Tube: 2000537
Kit: 1000688

Thaw at room temperature, vortex,
and centrifuge briefly.

−20°C

BeckmanCoulter
SPRIselectReagent-

Manufacturer’s recommendations.

-

Amp Mix B

Tube: 2000567
Kit: 1000668

Vortex, centrifuge briefly.

−20°C

QiagenBuffer EB-

10x
Magnetic
Separator

Component: 230003
or 2000431
Kit: 1000499

Tab 2 Text

TS Primer
Mix B

Place on ice

Obtain-See Tips & Best Practices.

Ambient

Prepare fresh. Prepare 1 ml per
reaction.

Ambient

Tab 3 Text

80% Ethanol

Ambient

Tab 4 Text
Tab 5 Text
Tab 6 Text

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5.1 Pre-Amplification
Tab 1 Text

a. Prepare Pre-Amplification Mix on ice according to the appropriate table.
Add reagents in the order listed. Pipette mix and centrifuge briefly.
Maintain on ice.
6.5 mm Slides
Pre-Amplification Mix

PN

Nuclease-free Water

1X
(µl)
19.5

2X +
10% (µl)

4X +
10% (µl)

42.9

85.8

2000567

25.0

55.0

110.0

TS Primer Mix B

2000537

2.5

5.5

11.0

-

47.0

103.4

206.8

Total

Tab 2 Text

Amp Mix B

b. Add 47 µl Pre-Amplification Mix to each tube from 4.3 Probe Elution on
page 76. Pipette mix and centrifuge briefly.

Reaction Volume

Run Time

105°C

100 µl~25 min

Step

Temperature

Time hh:mm:ss

1

98°C

00:03:00

2

98°C

00:00:15

363°C

00:00:20

472°C

00:00:30

5

Tab 4 Text

Lid Temperature

Tab 3 Text

c. Incubate in a thermal cycler with the following protocol.

Go to Step 2, repeat 9X for a total of 10 cycles

6

72°C

00:01:00

7

4°C

Hold

Tab 5 Text
Tab 6 Text

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5.2 Pre-Amplification Cleanup - SPRIselecta. Vortex to resuspend SPRIselect reagent. Add 120 µl SPRIselect reagent(1.2X) to each pre-amplification reaction in an 8-tube strip (100 µl) andpipette mix 15x (pipette set to 175 µl).

Tab 1 Text

If using MicroAmp 8-Tube Strips, use the high position (magnet•High) only
throughout the protocol.

b. Incubate 5 min at room temperature.

d. Remove supernatant.
e. Add 300 µl 80% ethanol to pellet. Wait 30 sec. Pipette carefully as 300 µl
is at tube limit.

Tab 2 Text

c. Place on the magnet•High until solution clears.

f. Remove ethanol.
g. Add 200 µl 80% ethanol to pellet. Wait 30 sec.

i. Centrifuge briefly and place on magnet•Low.
j. Remove any remaining ethanol without disturbing the beads. Air dry for 2
min. DO NOT exceed 2 min as this will decrease elution efficiency.

Tab 3 Text

h. Remove ethanol.

k. Remove from magnet. Add 105 µl Buffer EB. Pipette mix 15x (pipette set
to 100 µl).

Tab 4 Text

l. Incubate 2 min at room temperature.
m. Place tube strip on magnet •High until solution clears.
n. Transfer 100 µl sample to a new tube strip.
o. Store at 4°C for up to 72 h, -20°C for up to 4 weeks, or proceed to nextstep.

Tab 5 Text
Tab 6 Text

Step 5: Pre-Amplification and SPRIselect10xgenomics.com

80Step 6:
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6.0 Get Started

82

6.1 Cycle Number Determination – qPCR

83

6.2 Sample Index PCR

85

6.3 Post-Sample Index PCR Cleanup – SPRIselect87

6.4 Post-Library Construction QC

88

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6.0 Get Started
10x PN

Preparation & Handling

StorageTab 1 Text

Item

Equilibrate to room temperature
Tube: 2000537
Kit: 1000688

Thaw at room temperature,vortex, and centrifuge briefly.

−20°C

Dual Index
Plate TS Set A

Tube: 3000511
Kit: 1000251

Thaw at room temperature,vortex, and centrifuge briefly.

−20°C

BeckmanCoulter
SPRIselectReagent-

Manufacturer’s
recommendations.

-

Manufacturer’s
recommendations.

-

Agilent
TapeStation
Screen Tapeand Reagents
If used for QC

Tab 2 Text

TS Primer Mix
B-

Manufacturer’s
recommendations.

-

Amp Mix B

Component: 2000567
Kit:

Vortex, centrifuge briefly.
Maintain on ice.

−20°C

-

Manufacturer’s
recommendations.

-

Nuclease-free
Water

-

-

Ambient

Qiagen BufferEB-

10x Magnetic
Separator

Component: 230003 or
2000431
Kit: 1000499

See Tips & Best Practices.

Ambient

80% Ethanol-

Prepare fresh. Prepare 400 µl
per reaction.

Ambient

Tab 3 Text

Agilent
Bioanalyzer
High
Sensitivity
DNA kitIf used for QC
Place on ice

Tab 4 Text

KAPA SYBRFast qPCR
Master Mix
ObtainAmbient

Tab 5 Text
Tab 6 Text

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6.1 Cycle Number Determination – qPCR

b. Prepare qPCR Mix on ice according to the table below. Add reagents in theorder listed. Vortex and centrifuge briefly. Maintain on ice.

Stock

Final

1X
(µl)

3X* +
10% (µl)

5X* +10% (µl)

KAPA SYBR FASTqPCR Master Mix
Minimize light exposure

2X

1X

5.0

16.5

27.5

Diluted TS Primer Mix B

-

-

1.0

3.3

5.5

Nuclease-free Water

-

-

3.0

9.9

16.5

9.0

29.7

49.5

Total

Tab 3 Text

Gene Expression
qPCR Mix

Tab 2 Text

A passive reference dye, such as ROX, may be required. Consult the Roche
KAPA SYBR FAST qPCR Kit website or the appropriate qPCR systemmanufacturer for guidance.

Tab 1 Text

a. Dilute TS Primer Mix B 1:10 in nuclease-free water. The amount of diluted
primer mix required to generate the qPCR mixes is described in the tablebelow.

*Includes one negative control

c. Add 9 µl qPCR Mix to one well per sample in a qPCR plate (a well for
negative control may be included).

e. Transfer 1 µl diluted sample from Pre-Amplification Cleanup - SPRIselectto each qPCR plate well containing qPCR Mix. For the negative control,
add 1 µl nuclease-free water to the corresponding well. Pipette mix.

Tab 4 Text

d. Dilute 2 µl sample from Pre-Amplification Cleanup - SPRIselect in 8 µl
nuclease-free water. Pipette mix, centrifuge briefly.

f. Apply seal and centrifuge briefly. Record which sample is in which well ofthe qPCR plate.

Tab 5 Text
Tab 6 Text

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g. Prepare a qPCR system with the following protocol, place the plate in thethermal cycler, and start the program.
Reaction Volume

Run Time

105°C

10 µl

35 minStep

Temperature

Time hh:mm:ss

1

98°C

00:03:00

2

98°C

00:00:05

363°C

00:00:30

Tab 1 Text

Lid Temperature

Tab 2 Text

Read signal
4

Go to step 2, 24x (total of 25 cycles) -h. Record the Cq value for each sample.

If the Cq value is > 20 cycles, refer to Troubleshooting on page 101 or
contact support@10xgenomics.com before proceeding.

Tab 3 Text

Set the y-axis to a linear scale. Plot RFU on the y-axis if not using areference dye, or ∆Rn if using a reference dye. The threshold for
determining the Cq value should be set along the exponential phase of theamplification plot, at ~25% of the peak fluorescence value.

Representative qPCR Amplification Plots

Tab 4 Text
Tab 5 Text
Tab 6 Text

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6.2 Sample Index PCR

b. Prepare Amplification Master Mix and pipette mix 10x shortly before use.
Amplification Master Mix

Amp Mix B
Total

1X
(µl)

2X +10%(µl)

4X +10%
(µl)

-

45

99

198

2000567

25

55

110

-

70

154

308

Tab 2 Text

Nuclease-free Water

10x PN

Tab 1 Text

a. Choose the appropriate sample index sets to ensure that no sample indicesoverlap in a multiplexed sequencing run. Record the 10x Sample Index
name (PN-3000511 Plate TS Set A well ID) used.

c. Add 70 µl Amplification Master Mix to a tube in an 8-tube strip for eachsample.

e. Add 5 µl of an individual Dual Index TS Set A to each tube and record thewell ID used. Pipette mix and centrifuge briefly.
f. Incubate in a thermal cycler with the following protocol.

Reaction Volume

Run Time

105°C

100 µlVariable

Step

Temperature

Time hh:mm:ss

1

98°C

00:03:00

2

98°C

00:00:15

363°C

00:00:20

472°C

00:00:30

Go to step 2, use the Cq Value +1 as the total # of cycles. See table below for
total # of cycle examples.

6

72°C

00:01:00

7

4°C

Hold

Tab 5 Text5

Tab 4 Text

Lid
Temperature

Tab 3 Text

d. Add 25 µl of each sample from pre-amplification to a separate tube
previously aliquoted with Amplification Master Mix.

Round Cq values up to the nearest whole number and add one cycle(examples below). Samples within ±1 cycle numbers can be combined in asingle SI-PCR run at the higher cycle number.

Tab 6 Text

Do not combine samples if the cycle number difference is greater than 1 toavoid library overamplification.

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Example Cycle Numbers+1

Total Cycles

7.2

+1

9

8.5

+1

10

13.7

+1

15

Tab 1 Text

Cq Value from qPCR

Example Batched Cycles
+1

Total
Cycles

7.2

+1

9

9.5

+1

11

13.7

+1

15

14.6

+1

16

BatchedCyclesTab 2 Text

Cq Value from
qPCR

10

16

Any remaining pre-amplification material can be stored at 4°C for up to 72 h
or at -20°C for up to 4 weeks for generating additional libraries.

Tab 3 Text
Tab 4 Text
Tab 5 Text
Tab 6 Text

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6.3 Post-Sample Index PCR Cleanup – SPRIselectTab 1 Text

If using MicroAmp 8-Tube Strips, use the high position (magnet•High) only
throughout the protocol
a. Vortex to resuspend SPRIselect Reagent. Add 85 µl SPRIselect Reagent(0.85X) to each sample. Pipette mix 15x (pipette set to 175 µl).
b. Incubate 5 min at room temperature.
c. Place on magnet•High until solution clears.

Tab 2 Text

d. Remove supernatant.
e. Add 200 µl 80% ethanol to pellet. Wait 30 sec.
f. Remove ethanol.
g. Repeat steps e and f for a total of two washes.
h. Centrifuge briefly. Place on the magnet•Low.

j. Remove from magnet. Add 27 µl Buffer EB. Pipette mix 15x.

Tab 3 Text

i. Remove any remaining ethanol. Air dry for 2 min. DO NOT exceed 2 minas this will decrease elution efficiency.

k. Incubate 2 min at room temperature.
l. Place on the magnet•Low until solution clears.
m. Transfer 25 µl sample to a new tube strip.

Tab 4 Text

n. Store at -20°C for long-term storage.

Tab 5 Text
Tab 6 Text

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6.4 Post-Library Construction QC
b. Run 1 µl of sample on an Agilent Bioanalyzer High Sensitivity chip. If
peak is too small or flat, retry with a lower dilution. See Troubleshooting
on page 101 for more information.

Tab 1 Text

a. Dilute sample (1:50 dilution, i.e 1 µl sample in 49 µl of EB buffer) until it
is at an appropriate concentration for the Bioanalyzer.

Representative Trace
Tab 2 Text
Tab 3 Text

Alternate QC Methods:lAgilent TapeStationl

LabChip

Tab 5 Text

See Appendix on page 113 for representative trace

Tab 4 Text

Determine the average fragment size from the Bioanalyzer trace. The expected
average fragment size is 250 bp. This will be used as the insert size for libraryquantification.

See Post Library Construction Quantification on page 119

Tab 6 Text

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88Sequencing
Sequencing Libraries

90

Sequencing Depth

90

Sequencing Type & Run Parameters

97

Illumina Sequencer Compatibility

97

Sample Indices97

Library Loading

98

Sequencing Metrics

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Sequencing Libraries
Tab 1 Text

Libraries contain standard Illumina paired-end constructs, which begin with
P5 and end with P7. Spatial Barcodes are encoded at the start of TruSeq Read
1 (Read 1T), while i7 and i5 sample index sequences are incorporated as theindex read. Read 1T, and Small RNA Read 2 (Read 2S) are standard Illumina
sequencing primer sites used in paired-end sequencing. Read 1T are used tosequence the 43 bp Spatial Barcode and UMI. Read 2S is used to sequence the
Ligated Probe Insert. Sequencing these libraries produces a standard Illumina
BCL data output folder.

Tab 2 Text
Tab 3 Text

Sequencing Depth

For example, a Capture Area that is 60% covered tissue would require at least
165 million read pairs (0.60 X 275,000,000).

Tab 5 Text

10x Genomics recommends calculating the minimum sequencing depth forlibraries by multiplying the percentage of the Capture Area covered by tissue
by 275 million read pairs.

Tab 4 Text

The minimum sequencing depth for Visium HD is 275 million read pairs per
fully-covered Capture Area. The recommended minimum sequencing depth
was chosen because it achieved >50% sequencing saturation for >50% formalin
fixed, paraffin embedded (FFPE) tissues tested at 10x Genomics. To achieve>50% saturation for >50% of fresh frozen (FF) tissues tested by 10x Genomics,
700 million read pairs per fully-covered Capture Area were required. To
achieve >50% saturation for >50% of the Fixed Frozen (FxF) tissues tested by
10x Genomics, 500 million read pairs per fully-covered Capture Area wererequired.

Tab 6 Text

Sequencing

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Example: Sequencing Depth for a SamplelCalculate total sequencing
depth=(Coverage Area x 275,000,000
read pairslExample calculation for 60%
coverage:
0.60 x 275,000,000 read pairs =165,000,000 total read pairs forthat sample

Tab 2 Text

Estimate the approximate
Capture Area (%)
covered by the tissue section.

Tab 1 TextlSequencing

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Tab 6 Text

The second graph shows the relationship between sequencing depth andsequencing saturation for the same samples shown in the first graph. At the

Tab 5 Text

The figures below show the relationship between sequencing depth,
sequencing saturation, and mean UMIs per square µm across different tissuepreservation methods. The first graph shows the relationship between mean
UMIs per square µm and sequencing depth. At the same sequencing depth, FF
with good RNA quality will typically have higher UMIs per square µm
compared to FFPE. Around the recommended sequencing depth, the slope ofthe line representing the FFPE samples becomes smaller, indicating fastersaturation and smaller increases in UMIs per square µm with additionalsequencing.

Tab 4 Text

Visium HD library complexity, or the UMIs recovered from a tissue sample, isdependent on fixation method, tissue composition, tissue type, and RNA
quality. Visium HD FF libraries will typically be more complex compared toequivalent FFPE tissue libraries. At a particular sequencing depth, a FF tissue
run with Visium HD will typically result in a higher number of UMIs per
square µm and a lower sequencing saturation compared to an equivalent
FFPE sample of the same tissue type.

Tab 3 Text

Sequencing saturation, at a particular sequencing depth, is driven by thecomplexity of the library and the percent usable read pairs. To achieve
saturation with high complexity libraries (unique UMIs), more sequencing isrequired to read all unique UMIs present in the library. Saturation is achieved
more efficiently (with less read pairs) for libraries with a high percentage ofusable read pairs. The main driver of saturation for Visium HD libraries islibrary complexity.

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Tab 1 Text

same sequencing depth, FF with good RNA quality will typically have lower
saturation compared to FFPE due to higher complexity. In these cases,
additional sequencing is often desirable.

Tab 2 Text
Tab 3 Text-

Tab 4 Text
Tab 5 Text
Tab 6 Text

Sequencing

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Tissue Compositions Tested
Heterogenous - variety of cell types andstructures

Nuclei-Dense - cells are very compact

Sparse Coverage - cells are separated bylarge spaces

Tab 2 Text

Stroma-Rich - extracellular matrix, connective tissue, or
adipose tissue

Tab 1 Text

Tissue composition can also influence the ideal sequencing depth for asample. The figures below demonstrate the relationship between sequencing
depth and mean UMIs per square µm for a variety of sample types to help
guide selection of sequencing depth. All H&E images below were taken at
20X magnification.

Tab 3 Text
Tab 4 Text

Homogenous - uniform cell types and structures

Tab 5 Text
Tab 6 Text

Sequencing

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Tab 1 Text
Tab 2 Text
Tab 3 Text

The above graph shows the relationship between sequencing saturation per
sample and sequencing depth per sample.

Tab 4 Text
Tab 5 Text

Sequencing

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Tab 6 Text

The above graph shows the relationship between mean UMIs per square µm of
the Capture Area covered by tissue and sequencing depth per sample.

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Tab 1 Text
Tab 2 Text
Tab 3 Text

The above graph shows the relationship between sequencing saturation per
sample and sequencing depth per sample.

Tab 4 Text
Tab 5 Text

Sequencing

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Tab 6 Text

The above graph shows the relationship between mean UMIs per square µm of
the Capture Area covered by tissue and sequencing depth per sample.

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Tab 1 Text
Tab 2 Text
Tab 3 Text

The above graph shows the relationship between sequencing saturation per
sample and sequencing depth per sample.

Tab 4 Text
Tab 5 Text

Sequencing

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Tab 6 Text

The above graph shows the relationship between mean UMIs per square µm of
the Capture Area covered by tissue and sequencing depth per sample.

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Sequencing Type & Run Parameters
Tab 1 Text

Use the sequencing run type and parameters indicated.
Visium HD Spatial Gene Expression - Probe-based Library
Paired-end, dual indexed sequencing
Read 1: 43 cycles
i7 Index: 10 cycles
i5 Index: 10 cycles
Read 2S: 50 cycles

The compatibility of the listed sequencers has been verified by 10x Genomics.
Some variation in assay performance is expected based on sequencer choice.
NextSeq 2000

l

NovaSeq 6000

l

NovaSeq X Plus

Tab 3 TextlTab 2 Text

Illumina Sequencer Compatibility

Sample IndicesTab 5 Text

Color balance in the index read is important for optimal quality anddemultiplexing results. When pooling samples on the NovaSeq X Series
instruments, Illumina recommends avoiding index combinations which only
have signals from A bases, G bases, or G+A bases in any given cycle. Refer toIllumina’s guidance on index color balancing.

Tab 4 Text

Each well of the Dual Index Kit TS Set A (PN-1000251) contains a mix of one
unique i7 and one unique i5 sample index. If multiple samples are pooled in asequence lane, the sample index name (i.e. the Dual Index TS Set A plate well
ID, SI-TS-) is needed in the sample sheet used for generating FASTQs with“spaceranger mkfastq”. Samples utilizing the same sample index should not
be pooled together or run on the same flow cell lane, as this would not enable
correct sample demultiplexing.

Library Pooling

Sequencing

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Tab 6 Text

Visium HD libraries derived from samples prepared using different methods(such as FFPE, FF, FxF) may be pooled for sequencing, taking into accountdifferences in tissue covered spot on a Capture Area and per-spot read depth
requirements. Pooling Visium HD libraries with other 10x libraries forsequencing is not recommended.

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Library Loading

Instrument

Loading Concentration (pM)

PhiX (%)

650

1

NovaSeq 6000 Standard

100-150

1

NovaSeq 6000 XP Workflow

150-200

1

400

1

NextSeq 2000

NovaSeq X Plus

Tab 2 Text

Once quantified and normalized, libraries should be denatured and diluted asrecommended for Illumina sequencing platforms. Consult Illumina
documentation for denaturing and diluting libraries.

Tab 1 Text

Library quantification should be done with the KAPA DNA Quantification Kit
using the average insert size determined by Agilent Bioanalyzer, Perkin Elmer
LabChip, or Agilent Tapestation QC. Alternate methods to KAPA qPCR for
final library concentration may result in underquantification andconsequently overloading.

Tab 3 Text
Tab 4 Text
Tab 5 Text
Tab 6 Text

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Sequencing MetricslTargeted sequencing depth: Minimum of 275 million read pairs multipliedby fraction Visium HD slide tissue coverage.
Paired-end, dual indexing: Read 1: 43 cycles; i7 Index: 10 cycles; i5
Index: 10 cycles; Read 2: 50 cyclesllFinal libraries with fragment lengths within the expected size range foreach library type.
Reliable and accurate library quantification using the KAPA qPCR DNA
Quantification Kit for Illumina Platforms (Roche, KK4824) and the averageinsert size determined by Agilent Bioanalyzer, Perkin Elmer LabChip, orAgilent TapeStation QC. Alternative methods to KAPA qPCR, such as
Qubit, for final library quantification may result in underquantification
and result in overloading flow cells.
Sequencing platform loading concentrations follow recommendations
described in Library Loading on the previous page, which are based on
KAPA qPCR quantification. Overloading/over-clustering may result in poor
run performance, decrease sequencing quality, and lower total data output
as compared to optimally loaded runs. The loading recommendations for
an individual sequencer are listed as general guidance and additional
optimization may be required.

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Tab 6 Text

Sequencing

Generating sequencing data from sections that remain adhered to theslide.

Tab 5 TextlStarting with a high-quality tissue block.

Tab 4 TextlTab 3 Text

% Base by cycle and % ≥Q30 Quality Score distribution showed highly
consistent profiles for all sequencing platforms tested. The data serve as
guidelines for assessing the quality of library sequencing. Additional factorsthat may contribute to overall success of a sequencing run and impact
downstream application performance metrics include:lTab 2 TextlTab 1 Text

FFPE samples were processed according to documentation listed in the
Workflow Overview on page 13. Libraries were pooled and sequenced at theindicated run parameters to generate the % Base and % ≥Q30 plots shown in
the following figures. These same libraries were used to illustrate sequencecompatibility. All libraries were quantified with the KAPA DNA Quantification
Kit and sequenced with 1% PhiX. Though only FFPE libraries are shown inthis section, libraries from fresh frozen (FF) and fixed frozen (FxF) tissue
sections are expected to perform similarly. All libraries followed the following
sequencing configuration and run parameters:

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Probe-based Libraries
Tab 1 Text

Representative % Base and % ≥Q30 plots are from a pool of sixteen Probebased libraries sequenced on a NovaSeq 6000 SP flow cell.

Tab 2 Text

Instr.

Load
Conc.
(pM)

% Occupancy

%PF

R1

R2

R1

Yield per Lane
(Gb)

i7

i5

R2

GEX

Tab 3 Text

Libraries were sequenced on the NextSeq 2000, NovaSeq 6000 and NovaSeq X
Plus. "Data by Cycle" plots from the Illumina SAV software displaying thepercentage of base calls and Q30 quality scores are shown. Mapped Reads (%)
refers to Reads Mapped Confidently to Probe Set for Probe-based (GEX).

MappedReads(%)% ≥Q30

650

81.2

73.1

51.4

59.9

95.6

96.6

95.1

94.4

97.2

NovaSeq
6000 SP

300

93.2

82.8

135

160

95

92

89

93

92.1

NovaSeq X
Plus

400

84.7

71.79

41.4

48.3

95.9

94.6

94.7

95.3

97.7

Tab 4 Text

NextSeq2000

Tab 5 Text
Tab 6 Text

Sequencing

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Tab 1 Text

Before CytAssist Instrument Run
1. Number of Washes

Number of Washes

Fraction Reads Usable (Mean)

Post Hybridization Wash

1

0.29

2

0.41

3

0.79

1

0.69

2

0.75

Post Ligation Washes

Tab 3 Text

Wash

Tab 2 Text

Post hybridization and post ligation washes are critical for assay performance.
Failure to perform the correct number of washes can reduce the fraction oftargeted reads usable (see table below). A similar effect is observed when
washing for less than the recommended 5 min, or when reagent is carriedover during the washes. Remove all liquid from the well when washing, andrefer to User Guide for correct number of washes and incubation times.

2. Inadequate Visium HD Slide Preparationll

Failure to remove excess liquid from the back of the Visium HD Slide
before loading the slide onto the Visium CytAssist may result in distorted
fiducial imaging, which can impact fiducial registration.

Tab 4 Textl

Leaving Visium HD Slide in 0.1X SSC inside slide mailer longer than 4 hafter Visium HD Slide washing may result in spacer detachment.

Insufficient drying of Visium HD Slide prior to instrument run may resultin bubble entrapment and uneven flow of Probe Release Mix.

Tab 5 Text
Tab 6 Text

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During CytAssist Instrument Run
Tab 1 Text

1. Bubbles Trapped During Visium CytAssist Run
Bubbles may Result in no Usable Sequencing Reads during the CytAssist Assay
CytAssist ImageUMI Counts

Tab 2 Text
Tab 4 Text

Avoid generating bubbles during reagent dispensing by pipetting slowly andavoiding expelling air from the pipette tip. Additionally, ensure that the
frosted area of the tissue slides are outside of the alignment guide lines on
the Tissue Slide Stage and that tissue slides are compatible with the CytAssist
instrument. Consult the Visium HD Spatial Gene Expression Protocol Planner(CG000698) for information on tested slides.

Tab 3 Text

Bubbles during a sample run are rare, but may result in a lack of useable
reads in the tissue area where the bubbled occurred. The most common causeof bubbles is incomplete drying of the tissue slide and/or the Visium HD
slide. Bubbles can also result from inaccurate dispensing volume of Probe
Release Mix. Ensure that the entire well is inspected for bubbles, not just the
Capture Area. Ensure that the entire well is also dry during the drying step,
not just the Capture Area.

Tab 5 Text
Tab 6 Text

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2. Reagent Flow Failure
Tab 1 Text

Reagent Flow Failure May Cause Loss of Data
CytAssist ImageUMI CountTab 2 Text

Ensure that frosted areas of the tissue slide are not within the dashed zone on
the Visium Tissue Slide Stage. Keep Visium slides and tissue slides free fromphysical damage, dust, and debris. After Eosin wash, remove as much PBS as
possible by flicking the tissue slide and wiping any remaining droplets aroundtissue with a lint-free laboratory wipe.

Tab 3 Text

Inappropriate flow of reagents during a Visium CytAssist experiment run may
result in transcript mislocation. This may be caused by improper formulationof the Probe Release Mix or inaccurate loading volume onto the Visium
HD Slide. If the UMI map appears abnormal (complete loss of UMIs from asignificant portion of the tissue), contact support@10xgenomics.com.

Tab 4 Text

3. Visium CytAssist Overheating

Tab 5 Text

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Tab 6 Text

If the Visium CytAssist instrument overheats during an experiment run, therun will fail. A warning message will appear instructing users to carefully
remove slides and contact support@10xgenomics.com.

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After CytAssist Instrument Run

Upon opening the lid after a successful run, the Visium slide may pop out of
the grooves on the Visium Slide Stage. This does not impact assay
performance or slide quality.

Tab 1 Text

1. Slide Popped Off of Visium Slide StageTab 2 Text
Tab 3 Text

2. Visium HD Slide Removal Delayed
A delay in removing and processing the Visium HD Slide after run completion
may impact data quality.

Tab 4 Text

3. No qPCR Amplification
No amplification during Cycle Number Determination may be due to thefollowing:llIssues with pipetting small volumeslFailure to neutralize KOHlFailure to add primers to pre-amp or qPCR mixl

Tab 5 Text

Incorrect qPCR machine programming (i.e. detecting a fluorophore otherthan SYBR Green)

Unusually low recovery from tissue due to high percentage of connective
tissue in Capture Area
Incorrect preparation of Probe Release MixlMistake in Probe Release Mix addition timing

Tab 6 Textl10xgenomics.com

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Leakage from the Visium CytAssist Tissue Slide Cassette during workflow

Tab 1 Text
Tab 2 Text

4. Flat Line in Bioanalyzer Library Trace
A normal qPCR output but no peak visible in the Bioanalyzer trace may be
due to a mistake in the SI-PCR step, errors during SPRIselect cleanup, an
overdilution of the final library, Bioanalyzer issue, or thermal cycler failure.

Tab 3 Text
Tab 4 Text

Overloaded Trace

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Tab 6 Text

The image below is an example of an overloaded trace. Note the double peakat around 270 bp. The low and high molecular weight markers are in low
abundance compared to the sample. Ensure the library was diluted 1:50 prior
to loading per protocol recommendations. If the library was diluted, ensure
the library concentration is within the specification range of the automatedelectrophoresis kit being used. The library can be quantified using a Qubit
and diluted further if appropriate.

Tab 5 Text

5. Overloaded or Overamplified Trace

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Tab 1 Text
Tab 2 Text

Overamplified Trace
The image below is an example of an overamplified trace. Note peak at 400
bp.

Tab 3 Text
Tab 4 Text

For example, a sample with Cq value of 7.5 should go through 9 cycles during
SI-PCR (round up to 8 and add 1) and a sample with a Cq value of 9.2 should
go through 11 cycles during SI-PCR (round up to 10 and add 1). If running
together, samples should undergo 10 cycles during SI-PCR.

Tab 5 Text

Ensure interpretation of qPCR plot is correct. If necessary, batch samples into
separate SI-PCR runs if Cq is > 1 cycle. If batching samples, the cycle number
determined should be ±1. If needed, select the value in between.

Tab 6 Text

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Issues Impacting Tissue Analysis

Tab 2 Text

Tissue detachment may result in a lack of Fraction Reads Usable in the region
where detachment occurred. If the tissue has folded on itself, this may also
cause elevated UMI counts in the overlapping areas or a loss of spatiality due
to ineffective permeabilization. Tissue folding may also cause lower
UMI capture. Inspect images carefully to identify these areas. Ensure thatslides tested by 10x Genomics were used for tissue placement. For a list of
tested slides, refer to the Visium HD Spatial Gene Expression Application
Protocol Planner (CG000698). For more information, consult the sample
preparation documentation described in Workflow Overview.

Tab 1 Text

1. Tissue Detachment and Folding

Tissue Detachment May Cause a Loss of Gene Expression
CytAssist ImageUMI CountTab 3 Text
Tab 4 Text

Tissue Folding May Cause Elevated UMI Counts
CytAssist ImageUMI CountTab 5 Text
Tab 6 Text

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2. Tissue Not Within Allowable Area
Tab 1 Text

Tissue Outside of Allowable Area is Not Analyzed
Tissue Larger than Capture Area

Tissue Not Properly Aligned

Tab 2 Text

3. Tissue Segmentation Failure

Tab 3 Text

Tissues that are not placed within the allowable area on compatible glass
slides will not be analyzed. This may occur if the tissue is larger than the
Capture Area or if the tissue slide is not properly aligned when loading onto
the Tissue Slide Stage. Consult the Visium HD Protocol Planner (CG000698)
for information on tested glass slides.

Examples of Scenarios that Lead to Tissue Segmentation Failure
Tissue Composition/Morphology May Result in Inadequate Staining

Tab 4 Text
Tab 5 Text

Space Ranger may fail to detect tissue for a variety of reasons, which may
necessitate manual fiducial alignment and tissue detection via Loupe
Browser. Tissue segmentation failure may result from:l

If the contrast between background and tissue is poor (due to inadequate
staining, technical error, or tissue composition) tissue segmentation
failure may occur.

Tab 6 Text

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4. Tissue within Capture Area Not Analyzed
Tab 1 Text

Gasket Covers Portion of Tissue within Capture Area
H&E Stained Tissue

UMI Counts

Tab 2 Text

5. High Split-Mapped or Half-Mapped Reads

Tab 4 Text

Additionally, tissue placed within the Capture Area may be excluded from theassay workflow due to improper tissue slide alignment when loading thetissue slide into the Visium CytAssist instrument.

Tab 3 Text

If the gasket covers a portion of the tissue within the Capture Area, that area
will not be exposed to the appropriate reagents; thus, probes will not be
captured. In the example above, a lack of destaining on the left side of thetissue implies that this area was obscured by the gasket. Though the area iswithin the Capture Area, probe capture will not occur due to this obstruction.
Gasket obstruction may be caused by improper gasket assembly, resulting in
the gasket being placed directly onto areas of interest. In Loupe Browser,
manual alignment must be performed to exclude the tissue area obscured by
the gasket. Ensure that the area of interest is centered within the gasket.

Tab 5 Text

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Tab 6 Text

High split-mapped or half-mapped reads can result from insufficient and/or
inefficient washing of the tissue especially during the post-hybridization andpost-ligation wash steps, as well as poor tissue quality. Follow all bestpractices, including completely removing residual liquid prior to proceeding
to subsequent steps (e.g. using a P20 pipette to remove residual volume,
tilting the cassette to collect the liquid at a corner, see illustration below),User Guide | Visium HD Spatial Gene Expression Reagent Kits

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Tab 1 Text

performing the wash steps close to the thermal cycler, ensuring that thetissue section(s) do not dry out during the washes, checking that the cassetteis fully seated on the Thermocycler Adapter between performing each wash,
cassette is fully sealed when necessary to prevent evaporation, and ensuringthat the thermal cycler lid is set to the appropriate temperature to prevent
condensation during the incubation steps.

Tab 2 Text
Tab 3 Text

Damage to the hydrogel may result in data loss. Avoid damaging the hydrogel
by adhering to best practices when removing reagent from the well as
described in Reagent Addition to & Removal from Wells on page 25. In theexample below, a scratch on the hydrogel noted by the arrow has resulted in aloss of UMI count.

Tab 4 Text

6. Data Loss due to Hydrogel Damage

Hydrogel Damage May Result in Data Loss

Tab 5 Text

UMI CountTab 6 Text

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Tab 1 Text
Tab 2 Text
Tab 3 Text
Tab 4 Text
Tab 5 Text
Tab 6 Text

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Appendix
HD Workflow Overview for Processing 4 Tissue Slides with 1 Visium CytAssist
Instrument

114

HD Workflow Overview for Processing 4 Tissue Slides with 2 Visium CytAssist
Instruments

116

Post Library Construction Quantification

119

LabChip Traces

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Agilent TapeStation Traces

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Oligonucleotide Sequences

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To execute this workflow, two thermal cyclers are required. This table is meant as a reference only.
Consult the detailed steps in this user guide for execution.

Tab 1 Text

HD Workflow Overview for Processing 4 Tissue Slides with 1
Visium CytAssist Instrument

Tab 2 Text
Tab 3 Text
Tab 4 Text
Tab 5 Text
Tab 6 Text

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Tab 1 Text
Tab 2 Text
Tab 3 Text

HD Workflow Overview for Processing 4 Tissue Slides with 2
Visium CytAssist Instruments

Tab 4 Text

To execute this workflow, two thermal cyclers are required. This table is meant as a reference only.
Consult the detailed steps in this user guide for execution.

Tab 5 Text
Tab 6 Text

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Post Library Construction Quantificationb. Dilute 2 µl sample with deionized water to appropriate dilutions that fallwithin the linear detection range of the KAPA Library Quantification Kit
for Illumina Platforms. (For more accurate quantification, make thedilution(s) in duplicate).

Quantification Master Mix

1X (µl)SYBR Fast Master Mix + Primer

12

Water

4

Total

16

e. Add 4 µl sample dilutions and 4 µl DNA Standards to appropriate wells.
Centrifuge briefly.

Tab 3 Text

d. Dispense 16 µl Quantification Master Mix for sample dilutions and DNA
Standards into a 96 well PCR plate.

Tab 2 Text

c. Make enough Quantification Master Mix for the DNA dilutions per sample
and the DNA Standards (plus 10% excess) using the guidance for 1
reaction volume below.

Tab 1 Text

a. Thaw KAPA Library Quantification Kit for Illumina Platforms.

f. Prepare a qPCR system with the following protocol. Insert the plate andstart the program.
Run Time-

20 µl

35 minStep

Temperature

Time

1

95°C

00:03:00

295°C

00:00:05

367°C
Read signal

00:00:30

4

Go to Step 2, 29X (Total 30 cycles)

Tab 5 Text

Reaction Volume

Tab 4 Text

Lid Temperature

g. Follow the manufacturer’s recommendations for qPCR-based
quantification. For library quantification for sequencer clustering,
determine the concentration based on insert size derived from the
Bioanalyzer/TapeStation trace.

Tab 6 Text

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LabChip Traces
Tab 1 Text

Protocol Step 6.3 - GEX Post Library Construction QC
Representative Trace
Run manufacturer's recommended volume of diluted sample (1:5 dilution)

Tab 2 Text
Tab 3 Text

DNA High Sensitivity Reagent Kit was used. Protocol steps correspond to the steps in this User
Guide.

Tab 4 Text
Tab 5 Text
Tab 6 Text

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Agilent TapeStation Traces
Tab 1 Text

Agilent TapeStation High Sensitivity D5000 ScreenTape was used. Protocol
steps correspond to the steps in this User Guide.
Protocol Step 6.4 - GEX Post Library Construction QC
Representative Trace
Run manufacturer's recommended volume of diluted sample (1:20 dilution)

Tab 2 Text
Tab 3 Text
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Oligonucleotide Sequences
Tab 1 Text

Slide Primers
5'-TTTTTTTCCCTACACGACGCTCTTCCGATCT-UMI-SpatialBarcode-GVV-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3'

Tab 2 Text

Visium HD Spatial Gene Expression - Probe-based Library

3’-TTACTATGCCGCTGGTGGCTCTAGATGTG-N10-TGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA-UMI-SpatialBarcode-GVV-T30BN-Ligated_Probe_Insert-ACCTTAAGAGCCCACGGTTCCTTGAGGTCAGTG-N10-TAGAGCATACGGCAGAAGACGAAC-5’

Tab 3 Text

5’-AATGATACGGCGACCACCGAGATCTACAC-N10-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-UMI-SpatialBarcode-GVV-T30VN-Ligated_Probe_Insert-TGGAATTCTCGGGTGCCAAGGAACTCCAGTCAC-N10-ATCTCGTATGCCGTCTTCTGCTTG-3’

Tab 4 Text
Tab 5 Text
Tab 6 Text

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