CG000338 Rev F

USER GUIDE

Chromium
Next GEM
Single Cell Multiome
ATAC + Gene Expression
FOR USE WITH
Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle, 16 rxns PN-1000283, includes:
Chromium Next GEM Single Cell Multiome ATAC Kit A, 16 rxns PN-1000280
Chromium Next GEM Single Cell Multiome Reagent Kit A, 16 rxns PN-1000282
Library Construction Kit, 16 rxns PN-1000190

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle, 4 rxns PN-1000285, includes:
Chromium Next GEM Single Cell Multiome ATAC Kit A, 4 rxns PN-1000281
Chromium Next GEM Single Cell Multiome Reagent Kit A, 4 rxns PN-1000284
Library Construction Kit B, 4 rxns PN-1000279

Chromium Next GEM Chip J Single Cell Kit, 48 rxns PN-1000234
Chromium Next GEM Chip J Single Cell Kit, 16 rxns PN-1000230
Single Index Kit N Set A, 96 rxns PN-1000212
Dual Index Kit TT Set A, 96 rxns PN-1000215

Next GEM reagents are specific to Next GEM products and should not be used interchangeably with non-Next GEM reagents.
10xGenomics.com

Notices

NoticesDocument Number
CG000338 • Rev F

Legal Notices©2022 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 applicableterms and conditions of sale accompanying the purchase of such product. 10x Genomics provides nowarranty and hereby disclaims any and all warranties as to the use of any third-party products or protocols
described herein. The use of products described herein is subject to certain restrictions as set forth in theapplicable terms and conditions of sale accompanying the purchase of such product. A non-exhaustive
list of 10x Genomics’ marks, many of which are registered in the United States and other countries canbe viewed at: www.10xgenomics.com/trademarks. 10x Genomics may refer to the products or services
offered by other companies by their brand name or company name solely for clarity, and does not claim any
rights in those third-party marks or names. 10x Genomics products may be covered by one or more of thepatents as indicated at: www.10xgenomics.com/patents. The use of products described herein is subject
to 10x Genomics Terms and Conditions of Sale, available at www.10xgenomics.com/legal-notices, or suchother 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.
Notice to Customer: The listed products and their use are the subject of United States Patent Nos. 6,159,736,
and 6,294,385, European Patent No. 1115856 and related patents and patent applications licensed from the
Wisconsin Alumni Research Foundation.

Instrument & Licensed Software Updates Warranties
Updates to existing Instruments and Licensed Software may be required to enable customers to use
new or existing products. In the event of an Instrument failure resulting from an update, such failed
Instrument will be replaced or repaired in accordance with the 10x Limited Warranty, Assurance Plan orservice agreement, only if such Instrument is covered by any of the foregoing at the time of such failure.
Instruments not covered under a current 10x Limited Warranty, Assurance Plan or service agreement will
not be replaced or repaired.

Support
Email: support@10xgenomics.com
10x Genomics
6230 Stoneridge Mall Road
Pleasanton, CA 94588 USA

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Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

2

Document
Revision
Summary

Document Number

CG000338

Title

Chromium Next GEM Single Cell Multiome ATAC + Gene
Expression User Guide

Revision

Rev E to Rev F

Revision Date

August 2022

Specific Changes:• Updated pipette tip and thermal cycler recommendations.
• Updated chip assembly guidance in Tips & Best Practices and in step 2 - GEM Generation &Barcoding.
• Updated reagent volumes for preparing Elution Solution 1 in step 3.1f.
• Updated Troubleshooting section to include guidance on gasket misalignment and GEM transfer.

General Changes:• Updated for general minor consistency of language and terms throughout.

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Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

3

TOC

							
6
7
13
13
14
16

27
28
29
30
30

31
32
33
34
35
36
37
37

38
39
3.1 Post GEM Incubation Cleanup – Dynabeads
3.2 Post GEM Incubation Cleanup – SPRIselect40
42

Step 4 											Pre-Amplification PCR									

43
444.1 Prepare Pre-Amplification Mix
4.2 Pre-Amplification PCR
4.3 Pre-Amplification SPRI Cleanup

45
45
46

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

4

TOC

Step 5 											ATAC Library Construction								

48
495.1 Sample Index PCR
5.2 Post Sample Index Double Sided Size Selection – SPRIselect5.3 Post Library Construction QC

50
51
52

Step 6 											cDNA Amplification

53
54

6.1 cDNA Amplification
6.2 cDNA Cleanup – SPRIselect6.3 cDNA QC & Quantification

55
56
57

Step 7 											Gene Expression Library Construction						

58
59Step Overview (Step 7.1d)
7.1 Fragmentation, End Repair & A-tailing
7.2 Post Fragmentation, End Repair & A-tailing Double Sided Size Selection – SPRIselect7.3 Adaptor Ligation
7.4 Post Ligation Cleanup – SPRIselect
7.5 Sample Index PCR
7.6 Post Sample Index PCR Double Sided Size Selection – SPRIselect
7.7 Post Library Construction QC

60
61
62
63
64
65
66
67

Sequencing										Troubleshooting 										

71
72GEM Generation & Barcoding
Chromium Controller Errors

73
75

Appendix											

76Post Library Construction Quantification
Agilent TapeStation Traces
LabChip Traces
Assay Scheme Overview
Sequences

77
78
79
80
81

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

5

Introduction
Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagents
Chromium Accessories
Recommended Thermal Cyclers
Additional Kits, Reagents & Equipment
Protocol Steps & Timing
Stepwise Objectives

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Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

6

Introduction

Chromium Next GEM Single Cell Multiome Reagent Kit A, 16 rxns PN-1000282
Chromium Next GEM Single Cell
Multiome GEM Kit A, 16 rxns
PN-1000232 (store at −20°C)
Chromium
Next GEM Single Cell Multiome
GEM Kit A#

Store at -20°C

PN

Chromium Next GEM Single Cell
Multiome Amp Kit A, 16 rxns
PN-1000233 (store at −20°C)
Chromium
Next GEM Single Cell Multiome
Amp Kit A
Store at -20°C#

PN

Barcoding Reagent Mix

1 2000267

Pre-Amp Mix

1 2000270

Barcoding Enzyme Mix

1 2000266

Pre-Amp Primers

1 2000271

Template Switch Oligo

1 3000228

Amp Mix

2 2000047

Reducing Agent B

1 2000087

SI-PCR Primer B

1 2000128

Cleanup Buffer

2 2000088

cDNA Primers

1 2000089

Quenching Agent

1 2000269

10xGenomics.com

10xGenomics.com

Chromium Next GEM Single Cell
Multiome Gel Bead Kit A, 16 rxns
PN-1000231 (store at −80°C)
Chromium
Next GEM Single Cell Multiome
Gel Beads A
Store at -80°C#

Single Cell Multiome
Gel Beads A

Dynabeads™ MyOne™ SILANE,
PN-2000048 (store at 4°C)#

Dynabeads MyOne
SILANEPN

2 2000048

PN

2 2000261

10xGenomics.com

Click to TOC

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

7

Introduction

Chromium Next GEM Single Cell Multiome ATAC Kit A, 16 rxns PN-1000280 (store at −20°C)
Chromium
Next GEM Single Cell Multiome
ATAC Kit A
Store at -20°C#

PN

20X Nuclei Buffer

1 2000207

ATAC Buffer B

1 2000193

ATAC Enzyme B

1 2000265

10xGenomics.com

Library Construction Kit, 16 rxns PN-1000190 (store at −20°C)
Library Construction Kit
Store at -20°C#

PN

Fragmentation Enzyme

1 2000090

Fragmentation Buffer

1 2000091

Ligation Buffer

1 2000092

DNA Ligase

1 220110

Adaptor Oligos

1 2000094

Amp Mix

1 2000047

10xGenomics.com

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Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

8

Introduction

Chromium Next GEM Single Cell Multiome Reagent Kit A, 4 rxns PN-1000284
Chromium Next GEM Single Cell
Multiome GEM Kit A, 4 rxns
PN-1000236 (store at −20°C)
Chromium
Next GEM Single Cell Multiome
GEM Kit A#

Store at -20°C

PN

Chromium Next GEM Single Cell
Multiome Amp Kit A, 4 rxns
PN-1000237 (store at −20°C)
Chromium
Next GEM Single Cell Multiome
Amp Kit A
Store at -20°C#

PN

Barcoding Reagent Mix

1 2000267

Pre-Amp Mix

1 2000274

Barcoding Enzyme Mix

1 2000273

Pre-Amp Primers

1 2000271

Template Switch Oligo

1 3000228

Amp Mix

1 2000103

Reducing Agent B

1 2000087

SI-PCR Primer B

1 2000128

Cleanup Buffer

1 2000088

cDNA Primers

1 2000089

Quenching Agent

1 2000269

10xGenomics.com

10xGenomics.com

Chromium Next GEM Single Cell
Multiome Gel Bead Kit A, 4 rxns
PN-1000235 (store at −80°C)
Chromium
Next GEM Single Cell Multiome
Gel Beads A
Store at -80°C#

Single Cell Multiome
Gel Beads A

Dynabeads™ MyOne™ SILANE,
PN-2000048 (store at 4°C)#

Dynabeads MyOne
SILANEPN

1 2000048

PN

1 2000261

10xGenomics.com

Click to TOC

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

9Introduction

Chromium Next GEM Single Cell Multiome ATAC Kit A, 4 rxns PN-1000281 (store at −20°C)
Chromium
Next GEM Single Cell Multiome
ATAC Kit A
Store at -20°C#

PN

20X Nuclei Buffer

1 2000207

ATAC Buffer B

1 2000193

ATAC Enzyme B

1 2000272

10xGenomics.com

Library Construction Kit B, 4 rxns PN-1000279 (store at −20°C)
Library Construction Kit B
Store at -20°C#

PN

Fragmentation Enzyme

1 2000104

Fragmentation Buffer

1 2000091

Ligation Buffer

1 2000092

DNA Ligase

1 220131

Adaptor Oligos

1 2000094

Amp Mix

1 2000131

10xGenomics.com

Click to TOC

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

10

Introduction

Chromium Next GEM Chip J Single Cell Kit, 48 rxns PN-1000234(store at ambient temperature)
Chromium
Partitioning Oil
Partitioning Oil#

PN

Chromium
Recovery Agent
Recovery Agent

6 2000190

Chromium
Next GEM
Chip J & Gaskets

#

#

PN

6 220016

PN

Chromium Next GEM Chip J

6 2000264

Gasket, 6-pack

1 370017

10xGenomics.com

Chromium Next GEM Chip J Single Cell Kit, 16 rxns PN-1000230(store at ambient temperature)
Chromium
Partitioning Oil
Partitioning Oil#

PN

Chromium
Recovery Agent

2 2000190

Chromium
Next GEM
Chip J & Gaskets

Recovery Agent

#

#

PN

2 220016

PN

Chromium Next GEM Chip J

2 2000264

Gasket, 2-pack

1 3000072

10xGenomics.com

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Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

11

Introduction

Single Index Kit N Set A, 96 rxns PN-1000212 (store at −20°C)
Single Index Kit N Set A#

Single Index Plate N Set A

PN

1 3000427

Dual Index Kit TT Set A, 96 rxns PN-1000215 (store at −20°C)
Dual Index Kit TT Set A#

Dual Index Plate TT Set A

Click to TOC

PN

1 3000431

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

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Introduction

Chromium
Accessories

Recommended
Thermal Cyclers

Product

PN (Orderable)

PN (Item)

10x Vortex Adapter

120251330002Chromium Next GEM Secondary Holder

1000195

3000332

10x Magnetic Separator

120250230003

The table below lists the thermal cyclers that have been validated by 10x Genomics.
Thermal cyclers used must support uniform heating of 100 µl emulsion volumes.
Supplier

Description

Part Number

BioRadC1000 Touch Thermal Cycler
with 96-Deep Well Reaction
Module1851197

Analytik Jena†

Biometra TAdvanced 96 SG

846-x-070-241(x = 2 for 230 V; 4 for 115 V; 5 for 100 V,
50-60 Hz)

Eppendorf‡

Mastercycler X50s*

6311000010

Mastercycler Pro(discontinued)

North America 950030010
International 6321 000.019

Veriti 96-Well Thermal Cycler

4375786

Thermo Fisher
Scientific

For select instruments, ramp rates should be adjusted for all steps as described below:
Analytik Jena Biometra TAdvanced 96 SG: 2°C/sec for both heating and cooling†

Eppendorf Mastercycler X50s: 3°C/sec heating and 2°C/sec cooling‡

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Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

13

Introduction

Additional Kits,
Reagents &Equipment

The items in the table below have been validated by 10x Genomics and are highly
recommended for the Single Cell protocols. Substituting materials may adversely affectsystem performance. This list does not include standard laboratory equipment such as
water baths, centrifuges, vortex mixers, pH meters, freezers etc.

Supplier

Description

Part Number (US)PCR Tubes 0.2 ml 8-tube strips
DNA LoBind Tubes, 1.5 ml
DNA LoBind Tubes, 2.0 ml

951010022
022431021
022431048

Plastics
Eppendorf

Choose either
Eppendorf,
USA Scientific or
Thermo Fisher
Scientific PCR
8-tube strips.

USA Scientific

TempAssure PCR 8-tube strip

1402-4700

Thermo Fisher ScientificMicroAmp 8-Tube Strip, 0.2 ml
MicroAmp 8-Cap Strip, clear

RaininTips LTS 200UL Filter RT-L200FLR
Tips LTS 1ML Filter RT-L1000FLR
Tips LTS 20UL Filter RT-L10FLR

30389240
30389213
30389226

Thermo Fisher Scientific

Nuclease-free Water
Low TE Buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA)

AM993712090-015

Millipore Sigma

Ethanol, Pure (200 Proof, anhydrous)
Sigma Protector RNase Inhibitor
DTT

E7023-500ML
3335399001
646563Beckman Coulter

SPRIselect Reagent Kit

B23318

Bio-Rad

10% Tween 20

1662404

Ricca Chemical Company

Glycerin (glycerol), 50% (v/v) Aqueous Solution

3290-32

QiagenQiagen Buffer EB

19086

N8010580
N8010535

Kits & Reagents

Quantification & Quality Control
Agilent

2100 Bioanalyzer Instrument & Laptop Bundle
High Sensitivity DNA Kit
4200 TapeStation
High Sensitivity D1000 ScreenTape/ReagentsHigh Sensitivity D5000 ScreenTape/Reagents

Thermo Fisher Scientific

Qubit 4.0 Flourometer
Qubit dsDNA HS Assay Kit

Advanced Analytical

Fragment Analyzer Automated CE System - 12 cap
Fragment Analyzer Automated CE System - 48/96 cap
High Sensitivity NGS Fragment Analysis Kit

FSv2-CE2F
FSv2-CE10F
DNF-474

PerkinElmer

LabChip GX Touch HT Nucleic Acid Analyzer
DNA High Sensitivity Reagent Kit

CLS137031
CLS760672

KAPA Biosystems

KAPA Library Quantification Kit for Illumina Platforms

KK4824

Click to TOC

Choose Bioanalyzer,
TapeStation,
LabChip, Fragment
Analyzer or Qubit
based on availability& preference.

G2939BA & G2953CA
5067-4626
G2991AA
5067-5584/5067-5585
5067-5592/5067-5593
Q33226
Q32854Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

14

Introduction

Recommended
Pipette Tips

Supplier

10x Genomics recommends using only validated emulsion-safe pipette tips for all Single
Cell protocols. Rainin pipette tips have been extensively validated by 10x Genomics andare highly recommended for all single cell assays. If Rainin tips are unavailable, any of thelisted alternate pipette tips validated by 10x Genomics may be used.
Description

Part Number (US)Recommended Pipettes & Pipette tips
RaininPipettes
Pipet-Lite Multi Pipette L8-50XLS+
Pipet-Lite Multi Pipette L8-200XLS+
Pipet-Lite Multi Pipette L8-10XLS+
Pipet-Lite Multi Pipette L8-20XLS+
Pipet-Lite LTS Pipette L-2XLS+
Pipet-Lite LTS Pipette L-10XLS+
Pipet-Lite LTS Pipette L-20XLS+
Pipet-Lite LTS Pipette L-100XLS+
Pipet-Lite LTS Pipette L-200XLS+
Pipet-Lite LTS Pipette L-1000XLS+

17013804
17013805
1701380217013803
17014393
17014388
17014392
17014384
17014391
17014382

Pipette Tips
Tips LTS 200UL Filter RT-L200FLR
Tips LTS 1ML Filter RT-L1000FLR
Tips LTS 20UL Filter RT-L10FLR

30389240
30389213
30389226

Alternate Recommendations (If Rainin pipette tips are unavailable, any of the listed pipette tips may be used)
Pipettes
Eppendorf Research plus, 8-channel, epT.I.P.S. Box, 0.5 – 10 µL
Eppendorf Research plus, 8-channel, epT.I.P.S. Box, 10 – 100 µL
Eppendorf Research plus, 8-channel, epT.I.P.S. Box, 100 – 300 µL
Eppendorf Research plus, 1-channel, epT.I.P.S.® Box, 0.1 – 2.5 µL
Eppendorf Research plus, 1-channel, epT.I.P.S.® Box, 0.5 – 10 µL
Eppendorf Research plus, 1-channel, epT.I.P.S.® Box, 2 – 20 µL
Eppendorf Research plus, 1-channel, epT.I.P.S.® Box, 2 – 200 µL
Eppendorf Research plus, 1-channel, epT.I.P.S.® Box, 100 – 1000 µL

3125000010
3125000036
3125000052
3123000012
3123000020
3123000039
3123000055
3123000063

Pipette Tips (compatible with Eppendorf pipettes only)
ep Dualfilter T.I.P.S., 2-20 µL
ep Dualfilter T.I.P.S., 2-200 µL
ep Dualfilter T.I.P.S., 2-1,000 µL

0030078535
0030078551
0030078578

Labcon*ZAP SLIK 20 µL Low Retention Aerosol Filter Pipet Tips for Rainin LTS
ZAP SLIK 200 µL Low Retention Aerosol Filter Pipet Tips for Rainin LTS
ZAP SLIK 1000 µL Low Retention Aerosol Filter Pipet Tips for Rainin LTS

4-1143-965-008
4-1144-965-008
4-1145-965-008

Biotix*xTIP4 Racked Pipette Tips, Rainin LTS Pipette Compatible, 0.1-20uL
xTIP4 Racked Pipette Tips, Rainin LTS Pipette Compatible, 200uL
xTIP4 Racked Pipette Tips, Rainin LTS Pipette Compatible, 1000uL

63300931
63300001
63300003

Eppendorf*Compatible with Rainin pipettes

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Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

15

Introduction

Protocol Steps & Timing
Steps

TimingStop & Store

Nuclei Isolation
Dependent on Cell Type~1-2 hStep 1 – Transposition
1.1
1.2

Prepare Transposition Mix
Isothermal Incubation

10 min60 minStep 2 – GEM Generation & Barcoding
2.1
2.2
2.3
2.4
2.5
2.6

Prepare Master Mix
Load Chromium Next GEM Chip J
Run the Chromium Controller or X/iX
Transfer GEMs
GEM Incubation
Quenching Reaction

10 min10 min18 min3 min
75 min5 min

STOP−80°C ≤ 4 week

Step 3 – Post GEM Incubation Cleanup
3.1
3.2

Post GEM Incubation Cleanup – Dynabeads
Post GEM Incubation Cleanup – SPRIselect35 min15 minStep 4 – Library Pre-Amplification PCR
4.1
4.2
4.3

Prepare Pre-Amplification Mix
Pre-Amplification PCR
SPRI Cleanup

10 min30 min15 minSTOP

4°C ≤18 h
4°C ≤72 h or −20°C long-term

STOP

4°C ≤72 h or −20°C long-term

STOP

Step 5 – Single Cell ATAC Library Construction
5.1
5.2
5.3

Sample Index PCR
Post Sample Index Double Sided Size Selection –SPRIselectPost Library Construction QC

45 min20 min60 minStep 6 – cDNA Amplification
6.1
6.2
6.3

cDNA Amplification
cDNA Cleanup – SPRIselectcDNA QC & Quantification

40 min15 min50 minSTOP
STOP

4°C ≤72 h or −20°C ≤1 week
4°C ≤72 h or −20°C ≤4 weeks

Step 7 – Gene Expression Library Construction
7.1
7.2
7.3
7.4
7.5
7.6
7.7
Click to TOC

Fragmentation, End Repair & A-tailing
Post Fragmentation, End Repair & A-tailing Double
Sided – SPRIselectAdaptor Ligation
Post Ligation Cleanup- SPRIselectSample Index PCR
Post Sample Index PCR Double Sided – SPRIselectPost Library Construction QC

45 min30 min25 min20 min40 min STOP 4°C ≤72 h
30 min STOP 4°C ≤72 h or -20°C long term
50 minChromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

16

Introduction

Stepwise Objectives

Chromium Single Cell Multiome + Gene Expression provides a comprehensive,
scalable multiomic approach for simultaneously profiling epigenomic landscape andgene expression in the same single nuclei. This is achieved by transposing nuclei ina bulk solution. Using a microfluidic chip, the nuclei are partitioned into nanoliterscale Gel Beads-in-emulsion (GEMs). A pool of 736,000 10x Barcodes is sampled
to separately and uniquely index the transposed DNA and cDNA of each individual
nucleus. ATAC and gene expression (GEX) libraries are generated from the same poolof pre-amplified transposed DNA/cDNA and sequenced. The 10x Barcodes in eachlibrary type are used to associate individual reads back to the individual partitions,and thereby, to each individual nucleus.

Step 1
Transposition

Nuclei suspensions are incubated in a Transposition Mix that includes a Transposase.
The Transposase enters the nuclei and preferentially fragments the DNA in open
regions of the chromatin. Simultaneously, adapter sequences are added to the endsof the DNA fragments.

Step 2
GEM Generation &BarcodingSingle Cell Multiome ATAC + GEX Gel Beads include a poly(dT) sequence that enables
production of barcoded, full-length cDNA from poly-adenylated mRNA for gene
expression (GEX) library and a Spacer sequence that enables barcode attachment totransposed DNA fragments for ATAC library.

GEMs are generated by combining barcoded Gel Beads, transposed nuclei, a Master
Mix, and Partitioning Oil on a Chromium Next GEM Chip J. To achieve single nuclei
resolution, the nuclei are delivered at a limiting dilution, such that the majority (~9099%) of generated GEMs contains no nuclei, while the remainder largely contain asingle nucleus.

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Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

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Introduction

Upon GEM generation, the Gel Bead is dissolved. Oligonucleotides containing an
Illumina® P5 sequence, a 16 nt 10x Barcode (for ATAC), and a Spacer sequence arereleased. In the same partition, primers containing an Illumina® TruSeq Read 1 (read1 sequencing primer), 16 nt 10x Barcode (for GEX), 12 nt unique molecular identifier(UMI), and a 30 nt poly(dT) sequence are also released. The primers are mixed with
the nuclei lysate containing transposed DNA fragments, mRNA, and Master Mix, that
includes reverse transcription (RT) reagents.
Incubation of the GEMs produces 10x Barcoded DNA from the transposed DNA (forATAC) and 10x Barcoded, full-length cDNA from poly-adenylated mRNA (for GEX). Thisis followed by a quenching step that stops the reaction.
Inside individual GEMs

Step 3Post-GEM Cleanup

GEMs are broken and pooled fractions are recovered. Silane magnetic beads are used
to purify the cell barcoded products from the post GEM-RT reaction mixture, which
includes leftover biochemical reagents and primers.

Step 4Pre-Amplification PCR

Barcoded transposed DNA and barcoded full length cDNA from poly-adenylatedmRNA are amplified via PCR to fill gaps and for generating sufficient mass forlibrary construction. The pre-amplified product is used as input for both ATAC library
construction and cDNA amplification for gene expression library construction.
Pooled pre-amplification PCRTransposed DNA

Click to TOC

cDNA

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

18

Introduction

Step 5
ATAC Library
Construction

P7 and a sample index are added to pre-amplified transposed DNA during ATAC
library construction via PCR. The final ATAC libraries contain the P5 and P7
sequences used in Illumina® bridge amplification.
ATAC Library Construction

Step 6
cDNA Amplification

Barcoded, full-length pre-amplified cDNA is amplified via PCR to generate sufficientmass for gene expression library construction.

Step 7
Gene Expression Library
Construction

Enzymatic fragmentation and size selection are used to optimize the cDNA ampliconsize. P5, P7, i7 and i5 sample indexes, and TruSeq Read 2 (read 2 primer sequence) areadded via End Repair, A-tailing, Adaptor Ligation, and PCR. The final gene expression
libraries contain the P5 and P7 primers used in Illumina® bridge amplification.
cDNA Amplification &Gene Expression Library Construction

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Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

19

Introduction

Step 8
Sequencing

Chromium Single Cell Multiome ATAC libraries comprise double stranded DNA withstandard Illumina® paired-end constructs which begin with P5 and end with P7.
Sequencing these libraries produces a standard Illumina® BCL data output folder that
includes paired-end Read 1N and Read 2N used for sequencing the DNA insert, alongwith the 8 bp sample index in the i7 read and 16 bp 10x Barcode sequence in the i5
read.
Chromium Single Cell Multiome ATAC Library

Chromium Single Cell Multiome Gene Expression libraries comprise cDNA insert
with standard Illumina® paired-end constructs which begin with P5 and end with P7.
Sequencing these libraries produces a standard Illumina® BCL data output folder.
TruSeq Read 1 is used to sequence 16 bp 10x Barcodes and 12 bp UMI, while 10 bp i5and i7 sample index sequences are the sample index reads. TruSeq Read 2 is used tosequence the insert.

Chromium Single Cell Multiome Gene Expression Library

Click to TOC

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

20

Tips &
Best Practices
TIPS

Click to TOC

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

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Tips & Best Practices
TIPS

Icons
Tips & Best Practices
section includes
additional guidanceEmulsion-safe
Plastics

Multiplet Rate

GeneralReagent
Handling!
Signifies critical steprequiring accurate
execution

Troubleshooting section
includes additional
guidance• Use 10x Genomics validated emulsion-safe plastic consumables when handling
GEMs as some plastics can destabilize GEMs.

Multiplet Rate (%)# of Nuclei Loaded# of Nuclei Recovered

0.4%

~775

~500

0.8%

~1,550~1,0001.6%~3,075~2,0002.3%~4,625~3,000

3.1%~6,150~4,0003.9%~7,700~5,000

4.6%~9,250~6,0005.4%~10,750~7,0006.2%~12,300~8,0006.9%~13,850~9,0007.7%~15,400~10,000• Fully thaw and thoroughly mix reagents before use.
• Keep all enzymes and Master Mixes on ice during setup and use. Promptly movereagents back to the recommended storage.
• Calculate reagent volumes with 10% excess of reaction volumes.
• Cover Partitioning Oil tubes and reservoirs to minimize evaporation.
• Thoroughly mix samples with the beads during bead-based cleanup steps.

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Tips & Best Practices

50% Glycerol
Solution• Purchase 50% glycerol solution from Ricca Chemical Company, Glycerin (glycerol),50% (v/v) Aqueous Solution, PN-3290-32.
• Prepare 50% glycerol solution:i. Mix an equal volume of water and 99% Glycerol, Molecular Biology Grade.
ii. Filter through a 0.2 μm filter.
iii. Store at −20°C in 1-ml LoBind tubes. 50% glycerol solution should be equilibrated
to room temperature before use.

PipetteCalibration• Follow manufacturer’s calibration and maintenance schedules.
• Pipette accuracy is particularly important when using SPRIselect reagents.

Chromium
Next GEM Chip
Handling• Minimize exposure of reagents, chips, and gaskets to sources of particles and fibers,
laboratory wipes, frequently opened flip-cap tubes, clothing that sheds fibers, anddusty surfaces.
• After removing the chip from the sealed bag, use in ≤ 24 h.
• Execute steps without pause or delay, unless indicated. When multiple chips are to be
used, load, run, and collect the content from one chip before loading the next.
• Fill all unused input wells in rows labeled 1, 2, and 3 on a chip with an appropriate
volume of 50% glycerol solution before loading the used wells. DO NOT add glycerol
to the wells in the bottom NO FILL row.
• Avoid contacting the bottom surface of the chip with gloved hands and other surfaces.
Frictional charging can lead to inadequate priming of the channels, potentially leading
to either clogs or wetting failures.
• Minimize the distance that a loaded chip is moved to reach the Chromium Controller.
• Keep the chip horizontal to prevent wetting the gasket with oil, which depletes the
input volume and may adversely affect the quality of the resulting emulsion.

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23

Tips & Best Practices

Chromium Next GEM
Secondary Holders• Chromium Next GEM Secondary Holders
encase Chromium Next GEM Chips.
• The holder lid flips over to become a stand,holding the chip at 45 degrees for optimal
recovery well content removal.
• Squeeze the black sliders on the back sideof the holder together to unlock the lid andreturn the holder to a flat position.

Chromium Next GEM
Chip & Holder
Assembly with Gasket

Chromium Next GEM
Secondary Holder

Guide

Sliders

Clip• Close the holder lid. Attach the gasket by holding the tongue (curved end, to the right)
and hook the gasket on the left-hand tabs of the holder. Gently pull the gasket toward
the right and hook it on the two right-hand tabs.
• DO NOT touch the smooth side of the gasket.
• Open the chip holder.
• Align notch on the chip (upper left corner) and the open holder with the gasket
attached.
• Slide the chip to the left until the chip is inserted under the guide on the holder.
Depress the right hand side of the chip until the spring-loaded clip engages.
• Keep the assembled unit with the attached gasket until ready for dispensing reagents
into the wells.
Chip in Chromium Next GEM Secondary Holder

Chromium Next GEM
Secondary Holder
with Gasket

Assembled Chip

Notch
Guide
Clip

Chromium Next GEM
Chip Loading• Place the assembled chip and holder flat (gasket
attached) on the bench with the lid open.
• Dispense at the bottom of the wells without
introducing bubbles.
• When dispensing Gel Beads into the chip, wait for theremainder to drain into the bottom of the pipette tips
and dispense again to ensure complete transfer.
• Refer to Load Chromium Next GEM Chip J for
specific instructions.

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Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

24

Tips & Best Practices

Gel Bead
Handling

Lid
Gel Bead Strip
Holder• Use one tube of Gel Beads per sample. DO NOT
puncture the foil seals of tubes not used at the time.
• Equilibrate the Gel Beads strip to room
temperature before use.
• Store unused Gel Beads at −80°C and avoid
more than 12 freeze-thaw cycles. DO NOT
store Gel Beads at −20°C.

• Snap the tube strip holder with the Gel Bead strip into a 10x Vortex Adapter. Vortex 30 sec.
• Centrifuge the Gel Bead strip for ~5 sec after removing from the holder. Confirm
there are no bubbles at the bottom of the tubes and the liquid levels look even. Placethe Gel Bead strip back in the holder and secure the holder lid.
• If the required volume of beads cannot be recovered, place the pipette tips against the
sidewalls and slowly dispense the Gel Beads back into the tubes. DO NOT introduce
bubbles into the tubes and verify that the pipette tips contain no leftover Gel Beads.
Withdraw the full volume of beads again by pipetting slowly.

10x Magnetic
Separator• Offers two positions of the magnets (highand 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.
• If using MicroAmp 8-Tube Strips, use
the high position (magnet•High) only
throughout the protocol.

Magnetic Bead
Cleanup Steps• During magnetic bead based cleanup
steps that specify waiting “until thesolution clears”, visually confirm clearingof solution before proceeding to the next
step. See adjacent panel for an example.
• The time needed for the solution to clear
may vary based on specific step, reagents,
volume of reagents etc.

Magnetic beads mixed with reagent

Separation in

Separation

Separation complete; solution is

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Tips & Best Practices

SPRIselectCleanup &Size Selection• After aspirating the desired volume of SPRIselect reagent, examine the pipette tips
before dispensing to ensure the correct volume is transferred.
• Pipette mix thoroughly as insufficient mixing of sample and SPRIselect reagent willlead to inconsistent results.
• Use fresh preparations of 80% Ethanol.
Tutorial — SPRIselect Reagent:DNA Sample Ratios
SPRI beads selectively bind DNA according to the ratio of SPRIselect reagent (beads).
Example: Ratio = Volume of SPRIselect reagent added to the sample = 50 µl = 0.5X
			Volume of DNA sample

100 µlSchematic of Double Sided Size Selection

After the first SPRI, supernatant is transferred for a second SPRI while larger fragments are
discarded (green). After the second SPRI, fragments on beads are eluted and kept while smaller
fragments are discarded (blue). Final sample has a tight fragment size distribution with reduced
overall amount (black).

Tutorial — Double Sided Size Selection
Step a – First SPRIselect: Add 50 µl SPRIselect reagent to 100 µl sample (0.5X).
Ratio = Volume of SPRIselect reagent added to the sample = 50 µl = 0.5X
			Volume of DNA sample

100 µlStep b – Second SPRIselect: Add 30 µl SPRIselect reagent to supernatant from step a (0.8X).
Ratio = Total Volume of SPRIselect reagent added to the sample (step a + b) = 50 µl + 30 µl = 0.8X
			

Enzymatic
Fragmentation

Sample Indices in
Sample Index PCR

Original Volume of DNA sample		

100 µl• Ensure enzymatic fragmentation reactions are prepared on ice and then loaded into athermal cycler pre-cooled to 4°C prior to initiating the Fragmentation, End Repair, andA-tailing incubation steps.
• Choose the appropriate sample index sets to ensure that no sample indices overlap in
a multiplexed sequencing run.
• Each well in the Single Index plate N, Set A contains a unique mix of 4 oligos.
• Each well in the Dual Index Plate TT Set A contains a unique i7 and a unique i5
oligonucleotide.
• Verify and use the specified index plate only. DO NOT use the plates interchangebaly.

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26

Step 1
Transposition
1.1
1.2

Prepare Transposition Mix
Isothermal Incubation

1
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Step 1

1.0
Transposition

Transposition

GET STARTED!
ActionEquilibrate to
Room
Temperature

Item

10x PNATAC Buffer B

2000193

Vortex, centrifuge
briefly.

−20°C

20X Nuclei Buffer*

2000207

Thaw. Vortex,
centrifuge briefly.

−20°C

2000265/
2000272

Centrifuge briefly.

−20°C*Concentrated 20X
stock; dilute 1:20 innuclease-free water
before use. (See below
to Prepare Diluted
Nuclei Buffer)

Place on Ice

ATAC Enzyme B

Preparation & Handling

Storage

Nuclei**in Diluted Nuclei Buffer(See below to Prepare Diluted Nuclei Buffer)!

!

Prepare**Refer to Demonstrated Protocols for Nuclei Isolation for ATAC + Gene
Expression Sequencing (Documents CG000365, CG000366, CG000375,
CG000505). Adhering to these protocols is recommended for optimal assay
performance.

The use of the Tris-based Diluted Nuclei Buffer for nuclei suspension is criticalfor optimal assay performance. The composition of the Diluted Nuclei Buffer,including Magnesium concentration, has been optimized for the Transposition
and Barcoding steps. Suspension of nuclei in a different buffer may not becompatible with the downstream protocol steps.

DilutedNuclei
Buffer

Diluted Nuclei Buffer

Final

1 ml

20X Nuclei Buffer(PN-2000207)
DTT
RNase Inhibitor

20X

1X

50 µl

1,000 mM
40 U/µl

1 mM
1 U/µl

1 µl
25 µl

Nuclease-free Water--

924 µl(confirm vendor-specificstock concentration)

Click to TOC

Stock

Maintain at 4°C

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

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Step 1

Nuclei Concentration
Guidelines

Transposition
Based on the Targeted Nuclei Recovery, resuspend the nuclei in Diluted Nuclei Buffer to getcorresponding Nuclei Stock Concentrations (see Table). This enables pipetting volumes of the
Nuclei Stock for Transposition (step 1.1) to be 2-5 µl. Higher Nuclei Stock Concentrations will
result in lower pipetting volumes that may increase nuclei input variability.
Targeted Nuclei Recovery

Nuclei Stock Concentration(nuclei/µl)

500160-400

1,000

320-8102,000

650-1,610

3,000

970-2,420

4,000

1,290-3,230

5,000

1,610-4,030

6,000

1,940-4,840

7,000

2,260-5,650

8,000

2,580-6,450

9,000

2,900-7,260

10,0003,230-8,060

Calculate volume of Nuclei Stock and Diluted Nuclei Buffer for a total volume of 5 µl
Volume of Nuclei Stock (µl) = Targeted Nuclei Recovery x 1.61 (Recovery efficiency factor)
Nuclei Stock Concentration (nuclei/ µl)
Volume of Diluted Nuclei Buffer* (µl) = 5 µl - volume of Nuclei Stock (µl)*Use ONLY Diluted Nuclei Buffer (Dilute 20X Nuclei Buffer (PN-2000207) 1:20 in nuclease-free water)

Example Calculation
Targeted Nuclei Recovery = 4000 nuclei
Nuclei Stock Concentration = 2500 nuclei/µl
Recovery efficiency factor 1.61
Volume of Nuclei Stock (µl) =Targeted Nuclei Recovery x 1.61 (Recovery efficiency factor) = 4000 x 1.61 = 2.58 µl		Nuclei Stock Concentration (nuclei/µl)
2500
Volume of Diluted Nuclei Buffer = 5 µl - 2.58 µl = 2.42 µl
Add calculated volumes of Diluted Nuclei Buffer and Nuclei Stock to the Transposition Mix instep 1.1

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Step 1

Transposition

1.1
PrepareTransposition Mix

a. Prepare Transposition Mix on ice. Pipette mix 10x and centrifuge briefly.
Transposition Mix
Add reagents in the order listed

PN

1X (μl)4X +
10% (μl)8X +
10% (μl)

ATAC Buffer B

2000193

7.0

30.8

61.6

ATAC Enzyme B

2000265/
2000272

3.0

13.2

26.4

-

10.0

44.0

88.0

Total

b. Add 10 µl Transposition Mix to a tube of a PCR 8-tube strip for each sample.
Centrifuge briefly and maintain on ice.
c. Refer to Nuclei Concentration Guidelines to calculate the volume of Nuclei Stock andDiluted Nuclei Buffer for a total volume of 5 µl.
d. Add the calculated volume of Diluted Nuclei Buffer to the Transposition Mix. Pipettemix. Centrifuge briefly.

!
1.2
Isothermal Incubatione. Gently pipette mix the Nuclei Stock. Add the calculated volume of the Nuclei Stock to
the tube containing the Transposition Mix. Gently pipette mix 6x (pipette set to 10 µl).
DO NOT centrifuge.

a. Incubate in a thermal cycler using the following protocol.
Lid Temperature

Reaction Volume

Run Time
hh:mm:ss

50°C

15 µl

60 minStep

Temperature

Time

Incubate

37°C

00:60:00

Hold

4°C

Hold

b. Immediately proceed to the next step.

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Step 2
GEM Generation & Barcoding
2.1		
2.2		
2.3		
2.4
2.5		
2.6		

Prepare Master Mix
Load Chromium Next GEM Chip J
Run the Chromium Controller or X/iX
Transfer GEMs
GEM Incubation
Quenching Reaction

2
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Step 2

GEM Generation & Barcoding

2.0
GEM Generation &Barcoding

GET STARTED!
ActionEquilibrate to
Room
Temperature

Item

10x PN

Preparation & Handling

Storage

Single Cell Multiome
Gel Beads

2000261

Equilibrate to room
temperature 30 min
before loading the chip.

−80°C

Template Switch
Oligo

3000228

Centrifuge briefly,
resuspend in 80 µl Low
TE Buffer. Vortex 15 sec
at maximum speed,
centrifuge briefly, leaveat room temperaturefor ≥ 30 min. After
resuspension, store at−20°C−80°C. Thaw at room
temperature for ≥ 30
minutes in subsequent
uses.

Next GEM
specific!
Firmware Version 4.0 or higher
is required in the Chromium
Controller or the Chromium
Single Cell Controller used forthe protocol.

Reducing Agent B

2000087

Thaw, vortex, verify no
precipitate, centrifuge
briefly.

−20°C

Barcoding Reagent
Mix2000267

Thaw, vortex, verify no
precipitate, centrifuge
briefly.

−20°C

Place on Ice

Barcoding Enzyme
Mix

2000266/
2000273

Maintain on ice. Store at−20°C immediately after
use.

−20°C

ObtainPartitioning Oil

2000190-

Ambient

Low TE Buffer-

Manufacturer’s
recommendations.

-

Chromium Next GEM
Chip J

2000264

See Tips & Best Practices.

Ambient

10x Gasket

370017/3000072

See Tips & Best Practices.

Ambient

10x Vortex Adapter

330002

See Tips & Best Practices.

Ambient

Chromium Next GEM
Secondary Holder

3000332

See Tips & Best Practices

Ambient

50% glycerol
solution-

See Tips & Best Practices.

-

If using <8 reactions

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GEM Generation & Barcoding

Step 2

2.1
Prepare Master Mix

a. Prepare Master Mix on ice. Pipette mix 10x and centrifuge briefly.
Master Mix
Add reagents in the order listed

PN

1X (μl)4X +
10% (μl)8X +
10% (μl)

Barcoding Reagent Mix

2000267

49.5

217.8

435.6

Template Switch Oligo

3000228

1.1

4.8

9.7

Reducing Agent B

2000087

1.9

8.4

16.7

Barcoding Enzyme Mix

2000266/
2000273

7.5

33.0

66.0

-

60.0

264.0

528.0

Total

Assemble Chromium Next GEM Chip
TIPS

See Tips & Best Practices for chip handling instructions.
• Close the holder lid. Attach the gasket by holding the tongue (curved end, to the right)
and hook the gasket on the left-hand tabs of the holder. Gently pull the gasket toward theright and hook it on the two right-hand tabs.
• DO NOT touch the smooth side of the gasket.
• Open the chip holder.
• Remove the chip from the sealed bag. Use the chip within ≤ 24 h.
• Align notch on the chip (upper left corner) and the open holder with the gasket attached.
• Slide the chip to the left until the chip is inserted under the guide on the holder. Depress
the right hand side of the chip until the spring-loaded clip engages.
• Keep the assembled unit with the attached gasket open until ready for and while
dispensing reagents into the wells. DO NOT touch the smooth side of the gasket. Afterloading reagents, close the chip holder. DO NOT press down on the top of the gasket.
Chip in Chromium Next GEM Secondary Holder!
For GEM generation, load the indicatedreagents only in the specified rows,
starting from row labeled 1, followed
by rows labeled 2 and 3. DO NOT load
reagents in the bottom row labeled
NO FILL. See step 2.2 for details.

Click to TOC

Chromium Next GEM
Secondary Holder
with Gasket

Assembled Chip

Notch
Guide
Clip

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

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Step 2

2.2
Load Chromium
Next GEM Chip J

GEM Generation & Barcoding
After removing chip from the sealed bag, use in≤24 h. Open the lid (gasket attached) of the assembled
chip and lay flat for loading.
When loading the chip, raising and depressing thepipette plunger should each take ~5 sec.
When dispensing, raise the pipette tips at thesame rate as the liquid is rising, keeping the tips
slightly submerged.

a. Add 50% glycerol solution to each unused well

Glycerol(if processing <8 samples/chip)• 70 µl in each unused well in row labeled 1• 50 µl in each unused well in row labeled 2• 45 µl in each unused well in row labeled 3
DO NOT add 50% glycerol solution to the bottom row of NO FILL wells.
DO NOT use any substitute for 50% glycerol solution.

45 µl 350 µl 270 µl 1b. Prepare Master Mix + Cell suspension• Add 60 µl Master Mix to each tube containing Transposed Nuclei for a total of 75 µl in each tube.
c. Load Row Labeled 1• Gently pipette mix the Master Mix + Transposed Nuclei 5x• Using the same pipette tip, dispense 70 µl Master Mix + Transposed Nuclei into
the bottom center of each well in row labeled 1 without introducing bubbles.

Master Mix + Transposed Nuclei

70 µl 1NO FILL
The illustrated chip is being loaded for 8 samples.

d. Prepare Gel Beads• Snap the tube strip holder with the Gel Bead strip into a 10x Vortex Adapter.
Vortex 30 sec.
• Centrifuge the Gel Bead strip for ~5 sec.
• Confirm there are no bubbles at the bottom of the tubes and the liquid levels areeven.
• Place the Gel Bead strip back in the holder. Secure the holder lid.

Click to TOC

Prep Gel Beads

Lid
Gel Bead Strip
Holder

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GEM Generation & Barcoding

Step 2e. Load Row Labeled 2• Puncture the foil seal of the Gel Bead tubes.
• Slowly aspirate 50 µl Gel Beads.
• Dispense into the wells in row labeled 2 without introducing bubbles.
• Wait 30 sec.

Gel Beads

50 µl 2NO FILL

f. Load Row Labeled 3• Dispense 45 µl Partitioning Oil into the wells in row labeled 3 from a reagent
reservoir.
Failure to add Partitioning Oil to the top row labeled 3 will prevent GEM
generation and can damage the Chromium Controller or X/iX.

Partitioning Oil

45 µl 3
NO FILL

g. Prepare for Run• Close the lid (gasket already attached). DO NOT touch the smooth side of thegasket. DO NOT press down on the top of the gasket.
Run the chip in the Chromium Controller or X/iX immediately after loading thePartitioning Oil
Keep horizontal to avoid wetting thegasket. DO NOT press down on the gasket.

2.3
Run the Chromium
Controller or X/iX

If using Chromium Controller:a. Press the eject button on the Controller toeject the tray.

!

Firmware Version 4.00 or higher isrequired in the Chromium Controller or the
Chromium Single Cell Controller used forthe protocol.

b. Place the assembled chip with the gasket
in the tray, ensuring that the chip stays
horizontal. Press the button to retract the tray.
c. Press the play button.

!

Click to TOC

d. At completion of the run (~18 min), theController will chime. Immediately proceed
to the next step.

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

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GEM Generation & Barcoding

Step 2

If using Chromium X/iX:!

Consult the Chromium X
Series (X/iX) User Guide(CG000396) for detailed
instrument operation
instructions and follow the
instrument touchscreen
prompts for execution.

a. Press the eject button on Chromium X/iX toeject the tray.
If the eject button is not touched within 1 min,tray will close automatically. System requires
a few seconds before the tray can be ejectedagain.
b. Place the assembled chip with the gasket
in the tray, ensuring that the chip stays
horizontal Press the button to retract the
tray.
c. Press the play button.

!

d. At completion of the run (~18 min), Chromium
X/iX will chime. Immediately proceed to thenext step.

a. Place a PCR 8-tube strip on ice.

2.4
Transfer GEMs

b. Press the eject button of the Controller toremove the chip.

Expose Wells at 45 Degrees

c. Discard the gasket. Open the chip holder.
Fold the lid back until it clicks to expose thewells at 45 degrees.
d. Visually compare the remaining volume inrows labeled 1-2. Abnormally high volume in
one well relative to other wells may indicate
a clog.

Transfer GEMs

e. Slowly aspirate 100 µl GEMs from the lowest
points of the recovery wells in the top row
labeled 3 without creating a seal between
the pipette tips and the wells.

!

f. Withdraw pipette tips from the wells. GEMs
should appear opaque and uniform acrossall channels. Excess Partitioning Oil (clear) in
the pipette tips indicates a potential clog.

GEMs

g. Over the course of ~20 sec, dispense GEMs
into the tube strip on ice with the pipette tips
against the sidewalls of the wells.
h. If multiple chips are run back-to-back, cap/cover the GEM-containing tube strip or plate
and place on ice for no more than 1 h.
Click to TOC

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GEM Generation & Barcoding

Step 22.5
GEM Incubation

Use a thermal cycler that can accommodate at least 100 µl volume. A volume of
125 µl is the preferred setting on Bio-Rad C1000 Touch. In alternate thermal cyclers,
use highest reaction volume setting.
Incubate in a thermal cycler with the following protocol.
Lid Temperature

Reaction Volume

Run Time
hh:mm:ss

50°C

100 µl75 minStep

Temperature

Time

1

37°C

00:45:00

225°C

00:30:00

3

4°C

Hold
(not overnight*)!

Retrieve Quenching Agent ( PN-2000269) from -20°C and equilibrate to room
temperature while the PCR program is running.
*After GEM incubation, proceed immediately to the next step.

2.6
Quenching Reaction

a. Add 5 µl Quenching Agent to each sample to stop the reaction.

!
STOP

Click to TOC

b. Slowly pipette mix 10x (pipette set to 90 µl). The solution will be viscous.
Ensure that no liquid remains along the tube sidewalls and pipette tips. If necessary,
aspirate the entire volume and dispense back slowly into the tube.
c. Store at −80°C for up to 4 weeks, or proceed to the next step.

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

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Step 3
Post GEM Incubation Cleanup
3.1		
3.2		

Post GEM Incubation Cleanup – Dynabeads
Post GEM Incubation Cleanup – SPRIselect3
Click to TOC

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Step 3

3.0
Post GEM Incubation
Cleanup

Post GEM Incubation Cleanup

GET STARTED!
ActionItem

10x PN

Preparation & Handling

Storage

Reducing Agent B

2000087

Thaw, vortex, verify no
precipitate, centrifuge
briefly.

−20°C

Nuclease-free
Water

-

-

-

Dynabeads MyOne
SILANE

2000048

Vortex thoroughly(≥30 sec) to resuspend
beads immediately before
use.

4°C

Beckman Coulter
SPRIselectReagent-

Manufacturer’s
recommendations.

-Thaw at 65°C

Cleanup Buffer

2000088

Thaw for 10 min at
65°C at max speed on athermomixer. Verify there
are no visible crystals.
Cool to room temperature.

−20°C

ObtainRecovery Agent

220016-

Ambient

Qiagen Buffer EB-

Manufacturer’s
recommendations.

-

Bio-Rad 10%
Tween 20-

Manufacturer’s
recommendations.

-

10x Magnetic
Separator

230003-

Ambient

Prepare80% Ethanol-

Prepare fresh in nuclease
free water.

-

Equilibrate to
Room
Temperature

Prepare 10 ml for
8 reactions

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Post GEM Incubation Cleanup

Step 3a. Add 125 µl Recovery Agent to each sample
at room temperature. DO NOT pipette mix
or vortex the biphasic mixture. Gently invert
tube 10x to mix. Centrifuge briefly.

3.1
Post GEM Incubation
Cleanup – Dynabeads

Biphasic Mixture

The resulting biphasic mixture contains
Recovery Agent/Partitioning Oil (pink) andaqueous phase (clear), with no persistingemulsion (opaque).
A smaller aqueous phase volume indicates aclog during GEM generation.

!

Remove Recovery Agentb. Slowly remove and discard 125 µl Recovery
Agent/Partitioning Oil (pink) from the bottomof the tube. DO NOT aspirate any aqueous
sample.
c. Prepare Dynabeads Cleanup Mix.
Dynabeads Cleanup Mix
Add reagents in the order listed

PN

1X (μl)4X +
10% (μl)8X +
10% (μl)

2000088

182.0

801.0

1,602.0

Aspirate the full liquid volume witha pipette tip to verify that the beads
have not settled in the bottom of the
tube. If clumps are present, pipettemix to resuspend completely.
DO NOT centrifuge before use.

2000048

13.0

57.2

114.4

Reducing Agent B

2000087

5.0

22.0

44.0

-

200.0

880.2

1760.4

Cleanup Buffer
Dynabeads MyOne SILANE

Vortex thoroughly (≥30 sec)
immediately before adding to themix.

!
Resuspend
clump

Total

d. Vortex and add 200 µl Dynabeads Cleanup
Mix to each sample. Pipette mix 10x (pipette
set to 200 µl).

Add Dynabeads Cleanup Mix

e. Incubate 10 min at room temperature (keepcaps open).

Click to TOC

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

40

Post GEM Incubation Cleanup

Step 3f. Prepare Elution Solution I. Vortex and centrifuge briefly.
Elution Solution I*
Add reagents in the order listed

PN

1X (μl)4X +
15% (μl)8X +
15% (μl)

Buffer EB

-

98.0

450.8

901.6

10% Tween 20

-

1

4.6

9.2

Reducing Agent B

200087

1

4.6

9.2

-

100

460

920

Total
TIPS

g. At the end of 10 min incubation, place on the 10x Magnetic Separator, high position(magnet•High) until the solution clears.
h. Remove the supernatant.
i. Add 300 µl freshly prepared 80% ethanol to the pellet while on the magnet•High. Wait
30 sec.
j. Remove the ethanol.
k. Add 200 µl 80% ethanol to pellet. Wait 30 sec.
l. Remove the ethanol.
m. Centrifuge briefly. Place on the magnet•Low.
n. Remove remaining ethanol.
o. Remove from the magnet. Immediately add 50.5 µl Elution Solution I (prepared in step3.1f) to avoid clumping.
p. Pipette mix (pipette set to 40 µl) without introducing bubbles.
q. Incubate 1 min at room temperature.
r. Centrifuge briefly. Place on the magnet•Low until the solution clears.
s. Transfer 50 µl sample to a new tube strip.

Click to TOC

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

41

Step 3

Post GEM Incubation Cleanupa. Vortex the SPRIselect reagent until fully resuspended. Add 90 µl SPRIselect reagent
to each sample. Pipette mix thoroughly.

3.2
Post GEM Incubation
Cleanup – SPRIselectb. Incubate 5 min at room temperature.
c. Centrifuge briefly. Place on the magnet•High until the solution clears.
d. Remove the supernatant.
e. Add 200 µl 80% ethanol to the pellet. Wait 30 sec.
f. Remove the ethanol.
g. Repeat steps e and f for a total of 2 washes.
h. Centrifuge briefly. Place on the magnet•Low.

!

i. Remove any remaining ethanol.
Residual ethanol can inhibit Pre-Amplification PCR and impact assay performance.
j. Remove the tube strip from the magnet. Immediately add 46.5 µl Buffer EB.
k. Pipette mix (pipette set to 45 µl) without introducing bubbles.
l. Incubate 2 min at room temperature.
m.Centrifuge briefly. Place on the magnet•Low until the solution clears.

!

Click to TOC

n. Transfer 46 µl sample to a new tube strip.
Residual SPRI beads can inhibit Pre-Amplification PCR and impact assay performance.

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

42

Step 4Pre-Amplification PCR
4.1		
4.2		
4.3		

Prepare Pre-Amplification Mix
Pre-Amplification PCR
SPRI Cleanup

4
Click to TOC

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

43

Step 4Pre-Amplification PCR

4.0
Pre-Amplification PCR

GET STARTED!
ActionItem

10x PN

Preparation & Handling

Storage

Equilibrate to
Room
Temperature

Pre-Amp Primers

2000271

Vortex, centrifuge briefly.

−20°C

Beckman Coulter
SPRIselect Reagent-

Manufacturer’s
recommendations.

-

Place on Ice

Pre-Amp Mix

2000270/
2000274

Gently pipette mix,
centrifuge briefly.

−20°C

ObtainQiagen Buffer EB--

Ambient

10x Magnetic
Separator

230003

See Tips & Best Practices.

Ambient

Prepare80% Ethanol-

Prepare fresh.

Ambient

Next GEM
specific

Prepare 10 ml for 8
reactions

Click to TOC

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

44

Pre-Amplification PCR

Step 4

4.1
Prepare PreAmplification Mix

a. Prepare Pre-Amplification Mix on ice. Pipette mix 10x and centrifuge briefly.
Master Mix
Add reagents in the order listed

PN

1X (μl)4X +
10% (μl)8X +
10% (μl)

Pre-Amp Mix

2000270/
2000274

50.0

220.0

440.0

Pre-Amp Primers

2000271

4.0

17.6

35.2

-

54.0

237.6

475.2

Total

b. Add 54 μl Pre-Amplification Mix to each sample. Pipette mix and centrifuge briefly.

4.2
Pre-Amplification PCR

a. Incubate in a thermal cycler with the following protocol.
Lid Temperature

Reaction Volume

Run Time
hh:mm:ss

105°C

100 µl30 minStep

Temperature

Time

1

72°C

00:05:00

2

98°C

00:03:00

398°C

00:00:20

463°C

00:00:30

572°C

00:01:00
Go to step 3
repeat 6X (Total 7 cycles)

STOP

Click to TOC

6

72°C

00:01:00

7

4°C

Hold

b. Store at 4°C for up to 18 h or proceed to the next step.

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

45

Pre-Amplification PCR

Step 4a. Vortex the SPRIselect reagent until fully resuspended. Add 160 µl SPRIselect reagent
to each sample. Pipette mix thoroughly.

4.3
Pre-Amplification
SPRI Cleanupb. Incubate 5 min at room temperature.
c. Centrifuge briefly. Place on the magnet•High until the solution clears.
d. Remove the supernatant.
e. Add 300 µl 80% ethanol to the pellet. Wait 30 sec.
f. Remove the ethanol.
g. Add 200 µl 80% ethanol to the pellet. Wait 30 sec.
h. Remove the ethanol.
i. Centrifuge briefly. Place on the magnet•Low.
j. Remove any remaining ethanol.
k. Remove the tube strip from the magnet. Immediately add 160.5 µl Buffer EB.
l. Pipette mix (pipette set to 150 µl) without introducing bubbles.
m. Incubate 2 min at room temperature.
n. Centrifuge briefly. Place on the magnet•High until the solution clears.
o. Transfer 160 µl sample to a new tube strip.
STOP

Click to TOC

p. Store at 4°C for up to 72 h or at −20°C for long term storage, or proceed to the next
step.

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

46

Pre-Amplification PCR

Step 4

Sample Split
Overview• 160 µl pre-amplified, SPRI cleaned sample derived at step 4.3p includes barcodedtransposed DNA fragments and barcoded cDNA.
• The sample is divided and used as input for two separate steps.
I. 40 µl sample is used for ATAC Library Construction (step 5)
II. 35 µl sample is used for cDNA Amplification (step 6).
The amplified cDNA will be used for Gene Expression Library Construction.
Store the remaining pre-amplification product at −20°C long term for generating
additional libraries.

Pre-amplified sample (step 4.3p)
Barcoded transposed DNA

Barcoded cDNA

160 µl pre-amplified sample

40 µl sample

ATAC Library Construction
5.1b Sample Index PCR

Click to TOC

35 µl sample

cDNA Amplification
6.1b cDNA Amplification

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

47

Step 5
ATAC Library Construction
5.1		
5.2
5.3		

Sample Index PCR
Post Sample Index Double Sided Size Selection – SPRIselectPost Library Construction QC

5
Click to TOC

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

48

Step 5

5.0
ATAC Library
Construction

ATAC Library Construction

GET STARTED!
ActionEquilibrate to
Room
Temperature

Item

10x PN

Preparation & Handling

Storage

Sample Index
Plate N, Set A

3000427-−20°C

Beckman Coulter
SPRIselectReagent-

Manufacturer’s
recommendations.

-

Agilent
Bioanalyzer DNA
kit-

Manufacturer’s
recommendations.

-

DNA High
Sensitivity
Reagent Kit-

Manufacturer’s
recommendations.

-

SI-PCR Primer B

2000128

Vortex, centrifuge briefly.

−20°C

Amp Mix

2000047/
2000103

Vortex, centrifuge briefly.

−20°C

KAPA Library
Quantification Kit
for Illumina®
Platforms-

Manufacturer’s
recommendations.

-

Qiagen Buffer EB--

Ambient

10x Magnetic
Separator

230003

See Tips & Best Practices.

Ambient

Prepare80% Ethanol-

Prepare fresh.

Ambient

If used for QC

If LabChip used for
QC

Place on Ice

ObtainPrepare 10 ml
for 8 reactions

Click to TOC

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

49

Step 5

ATAC Library Construction

5.1
Sample Index PCR

Choose the appropriate sample index sets to ensure that no sample indicesoverlap in a multiplexed sequencing run.
a. Prepare Sample Index PCR Mix.
Sample Index PCR Mix
Add reagents in the order listed

PN

1X (μl)4X +
10% (μl)8X +
10% (μl)

Amp Mix

2000047/
2000103

50

220

440

SI- PCR Primer B

2000128

7.5

33

66

-

57.5

253

506

Total

b. Transfer 40 µl pre-amplified sample from step 4.3p to a new tube strip (35 µl of theremaining sample volume will be used for cDNA Amplification and the rest can be
stored at −20°C long term for generating additional libraries). Add 57.5 µl Sample
Index PCR Mix to the sample. Pipette mix and centrifuge briefly.
c. Add 2.5 µl of an individual Sample Index N, Set A to each well. Record assignment.
Pipette mix and centrifuge briefly.
d. Incubate in a thermal cycler with the following protocol.
Lid Temperature

Reaction Volume

Run Time
hh:mm:ss

105°C

100 µl~30 min

Step

Temperature

Time

1

98°C

00:00:45

2

98°C

00:00:20

367°C

00:00:30

472°C

00:00:20
Go to step 2, see table below
for # cycles

5

72°C

00:01:00

6

4°C

Hold

				
The table recommends a starting point
for cycle number optimization forbased on Targeted Nuclei Recovery.

STOP

Click to TOC

e. Store at 4°C for up to 72 h orproceed to the next step.

Cycle Number Optimization Table

Targeted Nuclei Recovery

Total Cycles≤2,00092,001-6,000

8

6,001-10,000

7Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

50

Step 5

ATAC Library Construction

5.2
TIPS
Post Sample Index
Double Sided Size
Selection – SPRIselecta. Vortex to resuspend SPRIselect reagent. Add 60 μl SPRIselect reagent (0.6X) to eachsample. Pipette mix.
b. Incubate 5 min at room temperature.
c. Place on the magnet•High until the solution clears.
d. Transfer 150 µl supernatant to a new strip tube. DO NOT discard the supernatant.
e. Vortex to resuspend SPRIselect reagent. Add 95 µl SPRIselect reagent (1.55X) to eachsample (supernatant). Pipette mix.
f. Incubate 5 min at room temperature.
g. Place on the magnet•High until the solution clears.
h. Remove the supernatant.
i. Add 300 µl 80% ethanol to the pellet. Wait 30 sec.
j. Remove the ethanol.
k. Add 200 µl 80% ethanol to the pellet. Wait 30 sec.
l. Remove the ethanol.
m. Centrifuge briefly. Place on the magnet•Low.
n. Remove remaining ethanol.
o. Remove from the magnet. Immediately add 20.5 µl Buffer EB. Pipette mix.
p. Incubate 2 min at room temperature.
q. Centrifuge briefly. Place on the magnet•Low until the solution clears.
r. Transfer 20 µl sample to a new tube strip.

STOP

Click to TOC

s. Store at 4°C for up to 72 h or at −20°C for long-term storage.

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

51

Step 5

5.3
Post Library Construction
QC

ATAC Library Construction

a. Run 1 μl sample on the Agilent Bioanalyzer High Sensitivity DNA chip to determine
fragment size. Select the region between 150-1000 bp to determine average size of
ATAC library. Lower molecular weight product (≤ 150 bp) and/or a high molecularweight product (~2,000 bp) may be present. This does not affect sequencing.
Representative Trace

Alternate QC Methods (See Appendix for representative traces)•

Agilent TapeStation•

LabChip
See Appendix for Post Library Construction Quantification

Click to TOC

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

52

Step 6
cDNA Amplification
6.1
6.2		
6.3

cDNA Amplification
cDNA Cleanup – SPRIselectcDNA QC & Quantification

6
Click to TOC

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

53

cDNA Amplification

Step 6

6.0
cDNA Amplification

GET STARTED!
ActionItem

10x PN

Preparation & Handling

Storage

cDNA Primers

2000089

Vortex, centrifuge briefly.

−20°C

Beckman Coulter
SPRIselectReagent-

Manufacturer’s
recommendations.

-

Agilent
Bioanalyzer HighSensitivity Kit-

Manufacturer’s
recommendations.

-

Agilent
TapeStation
ScreenTape andReagents-

Manufacturer’s
recommendations.

-

Qubit dsDNA HS
Assay Kit-

Manufacturer’s
recommendations.

-

Place on ice

Amp Mix

2000047/
2000103

Vortex, centrifuge briefly.

−20°C

ObtainQiagen Buffer EB-

Manufacturer’s
recommendations.

-

10x Magnetic
Separator

230003-

Ambient

Prepare 80% Ethanol

-

-

-

Equilibrate to
Room
Temperature

If used for QC andquantification

If used for QC andquantification

If used for QC andquantification

Prepare 15 ml
for 8 reactions.

Click to TOC

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

54

Step 6

cDNA Amplificationa. Prepare cDNA Amplification Mix on ice. Vortex and centrifuge briefly.

6.1
cDNA Amplification

cDNA Amplification Reaction Mix
Add reagents in the order listed

PN

1X (μl)4X +
10% (μl)8X +
10% (μl)

Amp Mix

2000047/
2000103

50

220

440

cDNA Primers

2000089

15

66

132

-

65

286

572

Total

b. Transfer 35 µl pre-amplified sample from step 4.3p to a new tube strip (store theremaining pre-amplification product at −20°C long term for generating additional
libraries). Add 65 µl cDNA Amplification Reaction Mix to the sample.
c. Pipette mix 15x (pipette set to 90 µl). Centrifuge briefly.
d. Incubate in a thermal cycler with the following protocol.
Lid Temperature

Reaction Volume

Run Time

105°C

100 µl~30-45 min

Step

Temperature

Time

1

98°C

00:03:00

2

98°C

00:00:15

363°C

00:00:20

472°C

00:01:00

5

Go to Step 2, see table below for total # of cycles

6

72°C

00:01:00

7

4°C

Hold

The optimal number of
				
				
Recommended starting point for cycle number optimization.
cycles is a trade-off between
generating sufficient final
mass for library construction
and minimizing PCRamplification artifacts. The
number of cDNA cycles
should also be reduced iflarge numbers of nuclei aresampled.

STOP

Click to TOC

Targeted Nuclei Recovery

Total Cycles≤2,00092,001–6,000

7≥6,001

6e. Store at 4°C for up to 72 h or or −20°C for ≤1 week, or proceed to the next step.

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

55

Step 6

cDNA Amplification & QC

a. Vortex to resuspend the SPRIselect reagent. Add 60 μl SPRIselect reagent (0.6X) toeach sample and pipette mix 15x (pipette set to 150 µl).

6.2
cDNA Cleanup –SPRIselectb. Incubate 5 min at room temperature.
c. Place on the magnet•High until the solution clears.
d. Remove the supernatant.
e. Add 200 µl 80% ethanol to the pellet. Wait 30 sec.
f. Remove the ethanol.
g. Repeat steps e and f for a total of 2 washes.
h. Centrifuge briefly and place on the magnet•Low.
i. Remove any remaining ethanol. Air dry for 2 min. DO NOT exceed 2 min as this will
decrease elution efficiency.
j. Remove from the magnet. Add 40.5 µl Buffer EB. Pipette mix 15x.
k. Incubate 2 min at room temperature.
l. Place the tube strip on the magnet•High until the solution clears.
m.Transfer 40 µl sample to a new tube strip.
STOP

Click to TOC

n. Store at 4°C for up to 72 h or at −20°C for up to 4 weeks, or proceed to the next step.

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

56

Step 6

6.3
cDNA QC & Quantification

cDNA Amplification & QC

a. Run 1 μl undiluted sample on an Agilent Bioanalyzer High Sensitivity chip.

For input cells with low RNA content (<1 pg total RNA/cell), 1 µl undiluted product may be run.
Lower molecular weight product (35 – 150 bp) may be present. This is normal and does not affect sequencing or
application performance.

Representative Trace

EXAMPLE CALCULATION
i. Select Region
Under the “Electropherogram” view choose the“Region Table”. Manually select the region of~200 – ~9000 bp

iii. Calculate
Multiply the cDNA concentration [pg/μl]
reported via the Agilent 2100 Expert Softwareby the elution volume (40 μl) of the Post cDNA
Amplification Reaction Clean Up sample (taking
any dilution factors into account) and then divideby 1000 to obtain the total cDNA yield in ng.
Example Calculation of cDNA Total Yield
Concentration: 6642.22 pg/µl
Elution Volume: 40
Dilution Factor: 1
Total cDNA Yield= Conc’n (pg/µl) x Elution Volume (µl) x Dilution Factor			1000 (pg/ng)

ii. Note Concentration [pg/µl]= 6642.22 (pg/µl) x 40 (µl) x 1 = 265.69 ng
		1000 (pg/ng)!

Carry forward ONLY 25% of total cDNA
yield into 3ʹ Gene Expression Library
Construction (step 3)= 0.25 x Total cDNA yield= 0.25 x 265.69= 66.42 ng

Refer to step 7.5 for appropriate number ofSample Index PCR cycles based on carryforward cDNA yield/input cDNA.

Alternate Quantification Methods (See Appendix for representative traces)•

Agilent TapeStation•

LabChip

Agilent Bioanalyzer, Agilent TapeStation, or LabChip are the recommended methods for
accurate quantification.
(If using Qubit Fluorometer and Qubit dsDNA HS Assay Kit, see Appendix)
Click to TOC

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

57

Step 7
Gene Expression Library Construction
7.1		
7.2
7.3		
7.4		
7.5		
7.6
7.7		

Fragmentation, End Repair & A-tailing
Post Fragmentation End Repair & A-tailing Double Sided Size Selection – SPRIselectAdaptor Ligation
Post Ligation Cleanup – SPRIselectSample Index PCR
Post Sample Index PCR Double Sided Size Selection – SPRIselectPost Library Construction QC

7
Click to TOC

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

58

Step 7

7.0

Gene Expression
Library Construction

Gene Expression Library Construction

GET STARTED!
ActionEquilibrate to
Room
Temperature

Item

10x PN

Preparation & Handling

Storage

Fragmentation
Buffer2000091

Vortex, verify no
precipitate, centrifuge
briefly.

−20°C

Adaptor Oligos

2000094

Vortex, centrifuge briefly.

−20°C

Ligation Buffer

2000092

Vortex, verify no
precipitate, centrifuge
briefly.

−20°C

Dual Index Plate TT
Set A

3000431-−20°C

Beckman Coulter
SPRIselect Reagent-

Manufacturer’s
recommendations.

-

Agilent TapeStation
Screen Tape andReagents

Manufacturer’s
recommendations.

If used for QC

Agilent Bioanalyzer
High Sensitivity kit-

Manufacturer’s
recommendations.

-

DNA High Sensitivity
Reagent Kit-

Manufacturer’s
recommendations.

-

Fragmentation
Enzyme2000090/2000104

Centrifuge briefly.

−20°C

DNA Ligase

220110/220131

Centrifuge briefly.

−20°C

Amp Mix

2000047/
2000131

Vortex, centrifuge briefly.

−20°C

KAPA Library
Quantification Kit
for Illumina
Platforms-

Manufacturer’s
recommendations.

-

If used for QC

If LabChip used for QC

Place on Ice

ObtainQiagen Buffer EB--

Ambient

10x Magnetic Separator

230003

See Tips & Best Practices.

Ambient

Prepare 80% Ethanol-

Prepare fresh.

Ambient

Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 User Guide • Rev A

59

Prepare 20 ml
for 8 reactions

Click to TOC

Step 7

Step Overview(Step 7.1d)

Gene Expression Library Construction

Correlation between input & library complexity
A Single Cell Gene Expression library is generated using a fixed proportion(10 µl, 25%) of the total cDNA (40 µl) obtained at step 6.2n. The complexity of thislibrary will be comparable to one generated using a higher proportion (>25%) of the
cDNA. The remaining proportion (30 µl, 75%) of the cDNA may be stored at 4°C for
up to 72 h or at −20°C for longer-term storage (up to 4 weeks).

Recommended(Median genes/UMI’s)

Library Complexity

Correlation: cDNA input & Library Complexity

cDNA Volume (µl)(% total cDNA)

10 µl
(25%)

20 µl
(50%)

30 µl
(75%)

40 µl
(100%)

Note that irrespective of the total cDNA yield (ng), which may vary based on cell
type, targeted nuclei recovery etc., this protocol has been optimized for a broad
range of input mass (ng), as shown in the example below. The total numberof SI PCR cycles (step 7.5d) should be optimized based on carrying forward afixed proportion (10 µl, 25%) of the total cDNA yield calculated during Post cDNA
Amplification QC & Quantification (step 6.3).

Example: Library Construction Input Mass & SI PCR Cycles
Cell TypeHigh RNA Content

Low RNA Content

Targeted
NucleiRecoverycDNA Input into
Fragmentation

SI PCR
Cycle
NumberVolume
(µl)

Mass
(ng)

150 ng10 µl37.5 ng

14High

400 ng10 µl

100 ng

13

Low

1 ng

10 µl

0.25 ng

16

100 ng

10 µl

25 ng

14

Low

High

Click to TOC

Total cDNA
Yield
(ng)

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

60

Step 7

Gene Expression Library Construction

7.1

a. Prepare a thermal cycler with the following incubation protocol.

Fragmentation,
End Repair & A-tailing!

Lid Temperature

Reaction Volume

Run Time
hh:mm:ss

65°C

50 µl

~35 min

Step

TemperatureTime

Pre-cool block

4°C

Hold

Fragmentation

32°C

00:05:00

End Repair & A-tailing

65°C

00:30:00

Hold

4°C

Hold

Pre-cool block prior to
preparing the Fragmentation Mix

b. Vortex Fragmentation Buffer. Verify there is no precipitate.
c. Prepare Fragmentation Mix on ice. Pipette mix and centrifuge briefly.
Fragmentation Mix
Add reagents in the order listed

PN

1X (μl)4X +
10% (μl)8X +
10% (μl)

Fragmentation Buffer

20000915

22

44

Fragmentation Enzyme

2000090/2000104

10

44

88

-

15

66

132

Total

d. Transfer ONLY 10 µl purified cDNA sample from cDNA Cleanup (step 6.2n) to a tube
strip.
Note that only 10 µl (25%) cDNA sample is sufficient for generating 3ʹ Gene Expression library.
The remaining 30 µl (75%) cDNA sample can be stored at 4°C for up to 72 h or at −20°C for up
to 4 weeks for generating additional Gene Expression libraries.

e. Add 25 µl Buffer EB to each sample.
f. Add 15 µl Fragmentation Mix to each sample.
g. Pipette mix 15x (pipette set to 35 µl) on ice. Centrifuge briefly.
h. Transfer into the pre-cooled thermal cycler (4°C) and press “SKIP” to initiate theprotocol.

Click to TOC

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

61

Step 7

7.2
Post Fragmentation,
End Repair & A-tailing
Double Sided Size
Selection – SPRIselectGene Expression Library Construction

a. Vortex to resuspend SPRIselect reagent. Add 30 μl SPRIselect (0.6X) reagent to eachsample. Pipette mix 15x (pipette set to 75 μl).
b. Incubate 5 min at room temperature.
c. Place on the magnet•High until the solution clears. DO NOT discard supernatant.
d. Transfer 75 μl supernatant to a new tube strip.
e. Vortex to resuspend SPRIselect reagent. Add 10 μl SPRIselect reagent (0.8X) to eachsample. Pipette mix 15x (pipette set to 80 μl).
f. Incubate 5 min at room temperature.
g. Place on the magnet•High until the solution clears.
h. Remove 80 μl supernatant. DO NOT discard any beads.
i. Add 125 μl 80% ethanol to the pellet. Wait 30 sec.
j. Remove the ethanol.
k. Repeat steps i and j for a total of 2 washes.
l. Centrifuge briefly. Place on the magnet•Low until the solution clears. Remove
remaining ethanol. Air dry for 2 min. DO NOT exceed 2 min as this will decrease
elution efficiency.
m. Remove from the magnet. Add 50.5 µl Buffer EB to each sample. Pipette mix 15x(pipette set to 45 µl) .
n. Incubate 2 min at room temperature.
o. Place on the magnet•High until the solution clears.
p. Transfer 50 μl sample to a new tube strip.

Click to TOC

Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide • Rev F

62

Step 77.3
Adaptor Ligation

Gene Expression Library Construction

a. Prepare Adaptor Ligation Mix. Pipette mix and centrifuge briefly.
Adaptor Ligation Mix
Add reagents in the order listed

PN

1X (μl)4X +
10% (μl)8X +
10% (μl)

Ligation Buffer

2000092

20

88

176

DNA Ligase

220110/220131104488Adaptor Oligos

2000094

20

88

176

-

50

220

440

Total

b. Add 50 µl Adaptor Ligation Mix to 50 µl sample. Pipette mix 15x (pipette set to 90 µl).
Centrifuge briefly.
c. Incubate in a thermal cycler with the following protocol.

Click to TOC

Lid Temperature

Reaction Volume

Run Time
hh:mm:ss

30°C

100 µl15 minStep

Temperature

Time

1

20°C

00:15:00

2

4°C

Hold

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Step 7

7.4
Post Ligation Cleanup –SPRIselect3ʹ Gene Expression Library Construction

a. Vortex to resuspend SPRIselect Reagent. Add 80 μl SPRIselect Reagent (0.8X) to eachsample. Pipette mix 15x (pipette set to 150 µl).
b. Incubate 5 min at room temperature.
c. Place on the magnet•High until the solution clears.
d. Remove the supernatant.
e. Add 200 µl 80% ethanol to the pellet. Wait 30 sec.
f. Remove the ethanol.
g. Repeat steps e and f for a total of 2 washes.
h. Centrifuge briefly. Place on the magnet•Low.
i. Remove any remaining ethanol. Air dry for 2 min. DO NOT exceed 2 min as this will
decrease elution efficiency.
j. Remove from the magnet. Add 30.5 µl Buffer EB. Pipette mix 15x.
k. Incubate 2 min at room temperature.
l. Place on the magnet•Low until the solution clears.
m. Transfer 30 µl sample to a new tube strip.

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Step 7

7.5
Sample Index PCR

3ʹ Gene Expression Library Construction

a. Choose the appropriate sample index sets to ensure that no sample indices overlap in
a multiplexed sequencing run. Record the 10x sample index name (PN-3000431 DualIndex Plate TT Set A well ID) used.

!

b. Add 50 µl Amp Mix (PN-2000047/2000131) to 30 µl sample.
c. Add 20 µl of an individual Dual Index TT Set A to each well and record the well ID used.
Pipette mix 5x (pipette set to 90 µl). Centrifuge briefly.
d. Incubate in a thermal cycler with the following protocol.
Lid Temperature

Reaction Volume

Run Time
hh:mm:ss

105°C

100 µl~25-40 min

Step

Temperature

Time

1

98°C

00:00:45

2

98°C

00:00:20

354°C

00:00:30

472°C

00:00:20

5

Go to step 2, see below for # of cycles

6

72°C

00:01:00

7

4°C

Hold

Recommended cycle numbers
The total cycles should be optimized basedon 25% carry forward cDNA yield/input
calculated during Post cDNA Amplification QC& Quantification (step 6.3)

!

STOP

Click to TOC

cDNA Input

Total Cycles

0.25-25 ng

14-16

25-150 ng

12-14

150-500 ng

10-12

500-1,000 ng

8-10

1,000-1,500 ng

6-8>1500 ng

5e. Store at 4°C for up to 72 h or proceed to the next step.

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Step 73ʹ Gene Expression Library Construction

7.6
Post Sample Index
PCR Double Sided Size
Selection – SPRIselecta. Vortex to resuspend the SPRIselect reagent. Add 60 μl SPRIselect Reagent (0.6X) toeach sample. Pipette mix 15x (pipette set to 150 μl).
b. Incubate 5 min at room temperature.
c. Place on the magnet•High until the solution clears. DO NOT discard supernatant.
d. Transfer 150 μl supernatant to a new tube strip.
e. Vortex to resuspend the SPRIselect reagent. Add 20 μl SPRIselect Reagent (0.8X) toeach sample (supernatant). Pipette mix 15x (pipette set to 150 μl).
f. Incubate 5 min at room temperature.
g. Place the magnet•High until the solution clears.
h. Remove 165 μl supernatant. DO NOT discard any beads.
i. With the tube still in the magnet, add 200 µl 80% ethanol to the pellet. Wait 30 sec.
j. Remove the ethanol.
k. Repeat steps i and j for a total of 2 washes.
l. Centrifuge briefly. Place on the magnet•Low. Remove remaining ethanol.
Air dry for 2 min. DO NOT exceed 2 min as this will decrease elution efficiency.
m. Remove from the magnet. Add 35.5 µl Buffer EB. Pipette mix 15x (pipette set to 35 µl).
n. Incubate 2 min at room temperature.
o. Place on the magnet•Low until the solution clears.
p. Transfer 35 µl to a new tube strip.

STOP

Click to TOC

q. Store at 4°C for up to 72 h or at −20°C for long-term storage.

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Step 7

7.7
Post Library Construction
QC

3ʹ Gene Expression Library Construction

Run 1 μl sample at 1:3 dilution on an Agilent Bioanalyzer High Sensitivity chip.
Representative Trace

Determine the average fragment size from the Bioanalyzer trace. This will be used as
the insert size for library quantification.
Alternate QC Methods (See Appendix for representative traces)•

Agilent TapeStation•

LabChip
See Appendix for Post Library Construction Quantification

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Sequencing

8
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Step 8

Sequencing Libraries

Sequencing

Chromium Single Cell Multiome ATAC libraries comprise double stranded DNA withstandard Illumina® paired-end constructs which begin with P5 and end with P7.
Sequencing these libraries produces a standard Illumina® BCL data output folder that
includes paired-end Read 1N and Read 2N used for sequencing the DNA insert, 8 bp
sample index in the i7 read, and 24 bp in the i5 read to cover the 16 bp 10x Barcodeand 8 bp Spacer.
Chromium Single Cell Multiome ATAC Library
SampleIndex (i5:16)

P5

SampleIndex N (i7:8)

Read 1N:50

10x Spacer
BarcodeRead 1N

InsertP7

Read 2N
Read 2N:49

Chromium Single Cell Multiome Gene Expression libraries comprise cDNA insert
with standard Illumina® paired-end constructs which begin with P5 and end with P7.
Sequencing these libraries produces a standard Illumina® BCL data output folder.
TruSeq Read 1 is used to sequence 16 bp 10x Barcodes and 12 bp UMI, while 10 bp i5and i7 sample index sequences are the sample index reads. TruSeq Read 2 is used tosequence the insert.
Chromium Single Cell Multiome Gene Expression Library
SampleIndex (i5:10)

P5

Illumina® Sequencer
Compatibility

Read 1:28
10x BC+UMI

TruSeq Read 1 10x
UMI
BarcodeSampleIndex (i7:10)

Poly(dT)VN

Read 2:90
Insert

TruSeq Read 2

P7

The compatibility of the listed sequencers has been verified by 10x Genomics. Somevariation in assay performance is expected based on sequencer choice. For more
information about performance variation, visit the 10x Genomics Support website.
• MiSeq™
• NextSeq™ 500/550 (High Output)• NextSeq™ 1000/2000• HiSeq 2500™ (Rapid Run)
• HiSeq™ 3000/4000• NovaSeq™

Sample Indices

Click to TOC

Each sample index in the Dual Index Kit TT Set A (PN-1000215) is a mix of one unique
i7 and one unique i5 sample index. Each i7 sample index in the Single Index Kit N Set
A (PN-1000212) is a mix of 4 different sequences to balance across all 4 nucleotides.
If multiple samples are pooled in a sequence lane, the sample index name (i.e. Single
Index Plate_ Set_well ID) is needed in the sample sheet used for generating FASTQs
with Cell Ranger. 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.
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Step 8ATAC Library Sequencing
Depth & Run Parameters

Sequencing!

These sequencing parameters are specific for Chromium Single Cell Multiome ATAC libraries
and are different from the recommendations for standalone ATAC libraries. Ensure that these
specific recommendations are provided to the sequencing service provider.

Sequencing Depth

25,000 read pairs per nucleus(25,000 reads for Read 1N; 25,000 reads for Read 2N)

Sequencing Type

Paired-end, dual indexing

Sequencing Read
Read 1N
i7 Index
i5 Index
Read 2N!

50 cycles
8 cycles24 cycles*
49 cycles*Custom sequencing recipe:
Sequencers that do not support 24 nt read in i5 (e.g. Nextseq™ 500/550) require a custom recipe
that includes 8 dark cycles and 16 nt cycles on i5. After installation of custom sequencing recipe,
input 16 cycles for i5 read.
It is imperative to use a custom recipe for these sequencers. Entering 16 cycles for i5 read
without the use of the custom recipe will cause the sequencing run to proceed but the data will
be unusable.
Custom recipe is NOT required for NextSeq™ 1000/2000.
Contact Support@10xgenomics.com for any additional questions.

ATAC Library Loading

Once quantified and normalized, ATAC libraries should be denatured and diluted
according to the table below. Consult the Technical Note on Sequencing Metrics andBase Composition of Single Cell Multiome ATAC Libraries (CG000373), available at the
10x Genomics Support website, for more information.
Instrument

Click to TOC

Loading Concentration (pM)

PhiX (%)

MiSeq™

10

1

NextSeq™ 500/550

1.5

1

NextSeq™ 1000/2000

650

1

HiSeq™ 2500 (RR)

10

1

HiSeq™ 4000

180

1

NovaSeq™

300

1

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Step 8Gene Expression Library
Sequencing Depth & Run
Parameters

Sequencing

Sequencing Depth

20,000 read pairs per nucleus

Sequencing Type

Paired-end, dual indexing

Sequencing Read

Recommended Cycles

Read 1i7 Index
i5 Index
Read 2

Gene Expression Library
Loading

28 cycles
10 cycles
10 cycles
90 cycles

Once quantified and normalized, libraries should be denatured and diluted according to
the table below. Refer to Illumina® documentation for denaturing and diluting libraries.
As the Multiome Gene Expression library is same as the Chromium Single Cell 3’ Gene
Expression Dual Index library, consult the Technical Note on Sequencing Metrics & BaseComposition of Single Cell 3’ v3.1 Dual Index Libraries (CG000374), available at the 10x
Genomics Support website, for more information.
Instrument

Loading Concentration (pM)

PhiX (%)

MiSeq™

11

1

NextSeq™ 500/550

1.8

1

NextSeq™ 1000/2000

650

1

HiSeq™ 2500 (RR)

11

1

HiSeq™ 4000

240

1

NovaSeq™

150*/300

1*Use 150pM loading concentration for Illumina XP workflow

Library Pooling

Click to TOC

Single Cell Multiome ATAC libraries maybe pooled with other ATAC libraries only whenusing forward strand Illumina® workflow. Single Cell Multiome Gene Expression libraries
maybe pooled for sequencing with other libraries, taking into account the differencesin cell number and per-cell read depth requirements between each library. 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. Refer to the 10x
Genomics Support website for more information.

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Troubleshooting

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Troubleshooting

GEM Generation &BarcodingSTEP

NORMAL

IMPACTED

2.2
Load Chromium
Next GEM Chip

Misaligned
gasketholes &
chip wells

Gasket holes are aligned with the sample
and gel bead wells.

2.4 d
After Chip J isremoved from theController or X/iX
and the wells are
exposed

A

B

C

D

E

F

G

Gasket holes are misaligned with the gelbead wells. Open and close the chip holder
slowly once.

A

H

All 8 recovery wells are similar in volume
and opacity.

B

C

D

E

F

G

H

Recovery well G indicates a reagent clog.
Recovery well C and E indicate a wetting
failure. Recovery wells B, D, and F arenormal. Wells A and H contain 50%
Glycerol Solution.

2.4 eTransfer GEMs
from Chip J
Row Labeled 3

Adequate emulsion volume(no clog or wetting failure)
Wetting failure
Low emulsion volume (clog)
A

All liquid levels are similar in volume andopacity without air trapped in the pipette
tips.

B

C

Pipette tip A shows normal GEM
generation, pipette tip B indicates a wetting
failure, and pipette tip C shows a clog andwetting failure.

Consult the Best Practices to Minimize Chromium Next GEM Chip Clogs and Wetting Failures (Technical Note CG000479) for more
information.

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Troubleshooting

STEP

NORMAL

IMPACTED

3.1 a
After transfer of the
GEMs +
Recovery Agent
A

B

C

D

E

F

G

A

H

All liquid levels are similar in the
aqueous sample volume (clear) andRecovery Agent/Partitioning Oil (pink).

B

C

D

E

F

G

H

Tube G indicates a reagent clog has
occurred. There is a decreased volume ofaqueous layer (clear).
Tube C and E indicate a wetting failure has
occurred. There is an abnormal volume of
Recovery Agent/Partitioning Oil (pink).

3.1 b
After aspiration of
Recovery Agent/Partitioning Oil
A

B

C

D

E

F

G

H

All liquid volumes are similar in the
aqueous sample volume (clear) and
residual Recovery Agent/Partitioning Oil(pink).

A

B

C

D

E

F

G

H

Tube G indicates a reagent clog has
occurred. There is a decreased volume
of aqueous layer (clear). There is also
a greater residual volume of Recovery
Agent/Partitioning Oil (pink).
Tube C and E indicate a wetting failure has
occurred. There is an abnormal residual
volume of Recovery Agent/Partitioning Oil(pink).

3.1 d
After addition of
Dynabeads
Cleanup Mix

A

B

C

D

E

F

G

H

All liquid volumes are similar after
addition of the Dynabeads Cleanup Mix.

A

B

C

D

E

F

G

H

Tube G indicates a reagent clog has
occurred. There is an abnormal ratio
of Dynabeads Cleanup Mix (brown) to
Recovery Agent/Partitioning Oil (appears
white).
Tube C and E indicate a wetting failure
has occurred. There is an abnormal ratio
of Dynabeads Cleanup Mix (brown) to
Recovery Agent/Partitioning Oil (appears
white).

If a channel clogs or wetting failure occurs during GEM generation, it isrecommended that the sample be remade.
If any of the listed issues occur, take a picture and send it to
support@10xgenomics.com for further assistance.

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Troubleshooting

Chromium Controller Errors
If the Chromium Controller or the Chromium Single Cell Controller fails to start, an error tone will sound and one of the following error messages will be
displayed:
a. Chip not read – Try again: Eject the tray, remove and/or reposition the Chromium Next GEM Secondary Holder assembly and try again. If the error message
is still received after trying this more than twice, contact support@10xgenomics.com for further assistance.
b. Check gasket: Eject the tray by pressing the eject button to check that the 10x Gasket is correctly installed on the Chromium Next GEM Chip. If the error
message persists, contact support@10xgenomics.com for further assistance.
c. Error Detected: Row _ Pressure:i. If this message is received within a few seconds of starting a run, eject the tray by pressing the eject button and check for dirt or deposits on the 10x
Gasket. If dirt is observed, replace with a new 10x Gasket, open and close the lid to ensure the gasket is properly aligned, and try again. If the error
message is still received after trying this more than twice, contact support@10xgenomics.com for further assistance.
ii. If this message is received after a few minutes into the run, the Chromium Next GEM Chip must be discarded. Do not try running this Chromium Next
GEM Chip again as this may damage the Chromium Controller.
d. Invalid Chip CRC Value: This indicates that a Chromium Next GEM Chip has been used with an older firmware version. The chip must be discarded. Contact
support@10xgenomics.com for further assistance.
e. Chip Holder Not Present: Open the controller drawer and check if chip holder is present. Insert chip properly into chip holder and retry.
f. Unauthorized Chip: This indicates that an incompatible non-Next GEM chip has been used with an instrument that only can run Next GEM assays. Use only
Chromium Controller (PN-120223;120246) or Chromium Single Cell Controller (PN-120263;120212) to run that chip or chip must be discarded. Contact
support@10xgenomics.com for further assistance.
g. Endpoint Reached Early: If this message is received, contact support@10xgenomics.com for further assistance.

Chromium X/iX Errors
The Chromium X touchscreen will guide the user through recoverable errors. If the error continues, or if the instrument has seen critical or intermediate
errors, email support@10xgenomics.com with the displayed error code. Support will request a troubleshooting package. Upload pertinent logs to 10x Genomics bynavigating to the Logs menu option on screen.
There are two types of errors:
Critical Errors — When the instrument has seen a critical error, the run will immediately abort. Do not proceed with any further runs. Contact support@10xgenomics.
com with the error code.
a.

System Errorb.

Pressure Errorc.

Chip Errord.

Run Errore.

Temperature Errorf.

Software Error
User Recoverable Errors — Follow error handling instructions through the touchscreen and continue the run.

a.

Gasket Errorb.

Tray Errorc.

Chip Errord.

Unsupported Chip Errore.

Update Error!

Consult the Chromium X Series (X/iX) User Guide (CG000396) for additional information and follow the Chromium X
touchscreen prompts for execution.

Click to TOC

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Appendix
Post Library Construction Quantification
Agilent TapeStation Traces
LabChip Traces
Assay Scheme Overview
Sequences

Click to TOC

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Appendix

Post Library Construction
Quantificationa. Thaw KAPA Library Quantification Kit for Illumina Platforms.
b. Dilute 2 µl sample with deionized water to appropriate dilutions that fall within thelinear detection range of the KAPA Library Quantification Kit for Illumina Platforms.
(For more accurate quantification, make the dilution(s) in duplicate).
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.
Quantification Master Mix

1X (µl)SYBR Fast Master Mix + Primer

12

Water

4

Total

16

d. Dispense 16 μl Quantification Master Mix for sample dilutions and DNA Standards into
a 96 well PCR plate.
e. Add 4 μl sample dilutions and 4 μl DNA Standards to appropriate wells. Centrifuge
briefly.
f. Incubate in a thermal cycler with the following protocol.
Step

Temperature

Run Time

1

95°C

00:03:00

295°C

00:00:05

367°C

00:00:30
Read signal

4

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

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.

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Appendix

Agilent TapeStation Traces
Agilent TapeStation Traces
Agilent TapeStation High Sensitivity D5000 ScreenTape was used.
Protocol steps correspond to the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide (CG000338).

Protocol Step 5.3 – Post Library Construction QC (ATAC Library)Run 2 μl sample

Protocol Step 6.3 – cDNA QC & Quantification

Run 2 μl sample

Protocol Step 7.7 – Post Library Construction QC (Gene Expression Library)Run 2 μl sample

All traces are representative.

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Appendix

LabChip Traces
LabChip Traces
DNA High Sensitivity Reagent Kit was used. Protocol steps correspond to the Chromium Next GEM Single Cell Multiome ATAC + GEX User Guide (CG000338).

Protocol Step 5.3 – Post Library Construction QC (ATAC Library)Run 10 µl undiluted sample

Protocol Step 6.3 – cDNA QC & Quantification

Run 10 µl undiluted sample

Protocol Step 7.7 – Post Library Construction QC (Gene Expression Library)Run 10 µl undiluted sample

All traces are representative.

Alternate QC Method:
Qubit Fluorometer and Qubit dsDNA HS Assay Kit. Multiply the cDNA concentration reported via the Qubit Fluorometer by theelution volume (40 µl) to obtain the total cDNA yield in ng. To determine the equivalent range using the Agilent 2100 Expert
Software, select the region encompassing 35-10,000 bp.
Click to TOC

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Appendix

Assay Scheme Overview

Click to TOC

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Appendix

Sequences
Single Cell Multiome Gel Beads A (PN- 2000261)
5’-CTACACGACGCTCTTCCGATCT-N16-N12-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3’
10x UMI
Read 1 Barcode
Poly(dT)VN
Gel
Bead
P5

10x Spacer
Barcode5’-AATGATACGGCGACCACCGAGATCTACAC-N16-CGCGTCTG-3’

Chromium Single Cell Multiome ATAC Library

P5

10x Spacer Read 1N
BarcodeInsertRead 2N Sample
Index NP7

5ʹ-AATGATACGGCGACCACCGAGATCTACAC-N16-CGCGTCTG-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG---insert--CTGTCTCTTATACACATCTCCGAGCCCACGAGAC-N8-ATCTCGTATGCCGTCTTCTGCTTG-3ʹ
3ʹ-TTACTATGCCGCTGGTGGCTCTAGATGTG-N16-GCGCAGAC-AGCAGCCGTCGCAGTCTACACATATTCTCTGTC---insert--GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG-N8-TAGAGCATACGGCAGAAGACGAAC-5ʹ

Chromium Single Cell Multiome Gene Expression Library
SampleIndex (i5)P5

10xBarcode

TruSeq Read 1

SampleIndex (i7)UMI

Poly(dT)VN

Insert TruSeq Read 2

P7

5’-AATGATACGGCGACCACCGAGATCTACAC-N10-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-N16-N12-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-cDNA_Insert-AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-N10-ATCTCGTATGCCGTCTTCTGCTTG-3’3’-TTACTATGCCGCTGGTGGCTCTAGATGTG-N10-TGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA-N16-N12-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAABN-cDNA_Insert-TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG-N10-TAGAGCATACGGCAGAAGACGAAC-5’

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