CG000168 Chromium Single Cell ATAC Reagent Kits User Guide Rev A
User Manual:
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Page Count: 47
- Introduction
- Chromium Single Cell ATAC Reagent Kits
- Chromium Accessories
- Recommended Thermal Cyclers
- Additional Kits, Reagents & Equipment
- Protocol Steps & Timing
- Stepwise Objectives
- Tips &Best Practices
- Step 1
- 1.0 Transposition
- Nuclei ConcentrationGuidelines
- 1.1 PrepareTransposition Mix
- 1.2 Isothermal Incubation
- Step 2
- 2.0 GEM Generation & Barcoding
- 2.1Prepare Master Mix
- 2.2Load Chromium Chip E
- 2.3Run the ChromiumController
- 2.4Transfer GEMs
- 2.5GEM Incubation
- Step 3
- 3.0Post GEM Incubation Cleanup & QC
- 3.2Post GEM Incubation Cleanup – SPRIselect
- Step 4
- 4.0Library Construction
- 4.1Sample Index PCR
- 4.2Post Sample Index Double Sided Size Selection – SPRIselect
- 4.3Post Library Construction QC
- 4.4Post Library Construction Quantification
- Sequencing
- Troubleshooting
- 6.1 GEMs
- 6.2 Chromium Controller Errors
10xGenomics.com
CG000168 Rev A
USER GUIDE
Chromium
Single Cell ATAC
Reagent Kits
FOR USE WITH
Chromium Single Cell ATAC Library & Gel Bead Kit, 16 rxns PN-1000110
Chromium Single Cell ATAC Library & Gel Bead Kit, 4 rxns PN-1000111
Chromium Chip E Single Cell ATAC Kit, 48 rxns PN-1000082 Chromium
Chip E Single Cell ATAC Kit, 16 rxns PN-1000086
Chromium i7 Multiplex Kit N, Set A, 96 rxns PN-1000084
2Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
Notices
Document Number
CG000168 | Rev A
Legal Notices
© 2018 10X Genomics, Inc. (10x Genomics). All rights reserved. Duplication and/or reproduction of all or
any portion of this document without the express written consent of 10x Genomics, is strictly forbidden.
Nothing contained herein shall constitute any warranty, express or implied, as to the performance of
any products described herein. Any and all warranties applicable to any products are set forth in the
applicable terms and conditions of sale accompanying the purchase of such product. 10x Genomics
provides no warranty and hereby disclaims any and all warranties as to the use of any third-party
products or protocols described herein. The use of products described herein is subject to certain
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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
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Email: support@10xgenomics.com
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Notices
3
Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
Table of Contents
Introduction 4
Chromium Single Cell ATAC Reagent Kits 5
Chromium Accessories 8
Recommended Thermal Cyclers 8
Additional Kits, Reagents & Equipment 9
Protocol Steps & Timing 10
Stepwise Objectives 11
Tips & Best Practices 13
Step 1 18
Transposition 19
Nuclei Concentration Guidelines 20
1.1 Prepare TranspositionMix 21
1.2 Isothermal Incubation 21
Step 2 22
GEM Generation & Barcoding 23
2.1 Prepare Master Mix 24
2.2 Load Chromium Chip E 25
2.3 Run the Chromium Controller 26
2.4 Transfer GEMs 26
2.5 GEM Incubation 27
Step 3 28
Post GEM Incubation Cleanup & QC 29
3.1 Post GEM Incubation Cleanup – Dynabeads 30
3.2 Post GEM Incubation Cleanup – SPRIselect 32
Step 4 33
Library Construction 34
4.1 Sample Index PCR 35
4.2 Post Sample Index Double Sided Size Selection – SPRIselect 36
4.3 Post Library Construction QC 37
4.4 Post Library Construction Quantification 38
Sequencing 39
Troubleshooting 42
6.1 GEMs 43
6.2 Chromium Controller Errors 45
Appendix 46
TOC
5Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
ATAC Buer 1 2000122
ATAC Enzyme 1 2000123
Nuclei Buer 1 2000153
Barcoding Reagent 1 2000124
Barcoding Enzyme 1 2000125
SI-PCR Primer B 12000128
Reducing Agent B 1 2000087
Amp Mix 1 2000047
Cleanup Buer 2 2000088
Chromium Single Cell ATAC Library Kit, 16 rxns PN-1000083 (store at −20°C)
Chromium
Single Cell ATAC
Library Kit
10xGenomics.com
#PN
Chromium Single Cell ATAC Gel Bead Kit, 16 rxns PN-1000081 (store at −80°C)
Chromium
Single Cell ATAC
Gel Beads
Single Cell ATAC
Gel Beads 2 2000132
10xGenomics.com
#PN
Dynabeads™ MyOne™ SILANE, PN-2000048 (store at 4°C)
Dynabeads MyOne
SILANE 1 2000048
#PN
Introduction
Chromium Single Cell ATAC Reagent Kits
Chromium Single Cell ATAC Library & Gel Bead Kit, 16 rxns PN-1000110
6Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
ATAC Buer 1 2000122
ATAC Enzyme 1 2000138
Nuclei Buer 1 2000153
Barcoding Reagent 1 2000124
Barcoding Enzyme 1 2000139
SI-PCR Primer B 12000128
Reducing Agent B 1 2000087
Amp Mix 1 2000103
Cleanup Buer 1 2000088
Chromium Single Cell ATAC Library Kit, 4 rxns PN-1000087 (store at −20°C)
Chromium
Single Cell ATAC
Library Kit
10xGenomics.com
#PN
Chromium Single Cell ATAC Gel Bead Kit, 4 rxns PN-1000085 (store at −80°C)
Chromium
Single Cell ATAC
Gel Beads
Single Cell ATAC
Gel Beads (4 rxns) 1 2000132
10xGenomics.com
#PN
Dynabeads™ MyOne™ SILANE, PN-2000048 (store at 4°C)
Dynabeads MyOne
SILANE 1 2000048
#PN
Introduction
Chromium Single Cell ATAC Reagent Kits
Chromium Single Cell ATAC Library & Gel Bead Kit, 4 rxns PN-1000111
7Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
#PN
10xGenomics.com
Chromium
Partitioning Oil
#PN
Chromium
Recovery Agent
Partitioning Oil 6 220088
Chromium
Chip E & Gaskets
Chip E Single Cell ATAC 6 2000121
Gasket, 6-pack 1 370017
#PN
Recovery Agent 6 220016
Chromium
Partitioning Oil
Partitioning Oil 2 220088
#PN
Chromium
Chip E & Gaskets
Chip E Single Cell ATAC 2 2000121
Gasket, 2-pack 1 3000072
#PN
10xGenomics.com
Chromium
Recovery Agent
Recovery Agent 2 220016
#PN
Chromium
i7 Multiplex Kit N
Set A
Chromium i7Sample Index
Plate N, Set A 1 3000262
#PN
Chromium Chip E Single Cell ATAC Kit, 48 rxns PN-1000082 (store at ambient temperature)
Chromium Chip E Single Cell ATAC Kit, 16 rxns PN-1000086 (store at ambient temperature)
Chromium i7 Multiplex Kit N, Set A, 96 rxns PN-1000084 (store at −20°C)
Introduction
8Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
Introduction
Recommended
Thermal Cyclers Supplier Description Part Number
BioRad C1000 Touch Thermal Cycler
with 96-Deep Well Reaction
Module
1851197
Eppendorf MasterCycler Pro North America 950030010
International 6321 000.01
Thermo Fisher
Scientific
Veriti 96-Well Thermal Cycler 4375786
Thermal cyclers used must support uniform heating of 100 µl emulsion volumes.
Chromium
Accessories Product PN (Orderable) PN (Item)
10x Vortex Adapter 120251 330002
10x Chip Holder 120252 330019
10x Magnetic Separator 120250 230003
9Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
The items in the table below have been validated by 10x Genomics and are highly
recommended for the Chromium Single Cell ATAC protocol. Substituting materials may
adversely aect system performance.
Additional Kits,
Reagents &
Equipment
Supplier Description Part Number (US)
Plastics
Eppendorf PCR Tubes 0.2 ml 8-tube strips
DNA LoBind Tubes, 1.5 ml
DNA LoBind Tubes, 2.0 ml
951010022
022431021
022431048
USA Scientific TempAssure PCR 8-tube strip 1402-4700
Rainin Tips LTS W-O 200UL Filter RT-L200WFLR
Tips LTS 20UL Filter RT-L10FLR
Tips LTS 200UL Filter RT-L200FLR
Tips LTS 1ML Filter RT-L1000FLR
30389241
30389226
30389240
30389213
Kits & Reagents
Thermo Fisher Scientific Nuclease-free Water AM9937
Corning Cellgro Phosphate-Buered Saline (PBS) 1X without calcium and magnesium 21-040-CV
Millipore Sigma Ethanol, Pure (200 Proof, anhydrous) E7023-500ML
Beckman Coulter SPRIselect Reagent Kit B23317
Bio-Rad 10% Tween 20 1662404
Ricca Chemical Company Glycerin (glycerol), 50% (v/v) Aqueous Solution 3290-32
Qiagen Qiagen Buer EB 19086
Equipment
Rainin Pipet-Lite Multi Pipette L8-50XLS
Pipet-Lite Multi Pipette L8-200XLS
17013804
17013805
VWR Vortex Mixer
Divided Polystyrene Reservoirs
10153-838
41428-958
Eppendorf ThermoMixer C 5382000015
Quantification & Quality Control
Agilent 2100 Bioanalyzer Laptop Bundle
High Sensitivity DNA Kit
4200 TapeStation
High Sensitivity D1000 ScreenTape
High Sensitivity D1000 Reagents
G2943CA
5067-4626
G2991AA
5067-5592
5067-5593
KAPA Biosystems KAPA Library Quantification Kit for Illumina® Platforms KK4824
Choose either
Eppendorf or
USA Scientific
PCR 8-tube
strips.
Choose
Bioanalyzer or
TapeStation,
based on
availability and
preferences.
Introduction
10Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
Steps Timing Stop & Store
3 h
Nuclei Isolation
Dependent on Cell Type ~1-2 h
Step 1 – Transposition
1.1
1.2
Prepare Transposition Mix
Isothermal Incubation
10 min
60 min
Step 2 – GEM Generation & Barcoding
2.1
2.2
2.3
2.4
2.5
Prepare Master Mix
Load Chromium Chip E
Run the Chromium Controller
Transfer GEMs
GEM Incubation
10 min
10 min
7 min
3 min
45 min
Step 3 – Post GEM Incubation Cleanup & QC
3.1
3.2
Post GEM Incubation Cleanup – Dynabeads
Post GEM Incubation Cleanup – SPRIselect
35 min
15 min 4°C ≤2 h or −20°C ≤ 2 weeks
Step 4 – Library Construction
4.1
4.2
4.3
Sample Index PCR
Post Sample Index Double Sided Size Selection –
SPRIselect
Post Library Construction QC
45 min
20 min
60 min
4°C ≤72 h or −20°C long-term
2 h
4 h
6 h
STOP
STOP
Introduction
Protocol Steps & Timing
11Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
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 ends of
the DNA fragments.
Stepwise Objectives
Step 1
Transposition
Step 2
GEM Generation &
Barcoding
The Chromium Single Cell ATAC Solution provides a comprehensive, scalable approach
to determine the regulatory landscape of chromatin in hundreds to thousands of cells
in a single sample. This is achieved by transposing nuclei in a bulk solution; then using a
microfluidic chip, the nuclei are partitioned into nanoliter-scale Gel Beads-in-emulsion
(GEMs). GemCode Technology samples a pool of ~750,000 10x Barcodes to separately
and uniquely index the transposed DNA of each individual cell. Libraries are generated
and sequenced, and 10x Barcodes are used to associate individual reads back to the
individual partitions, and thereby, to each individual cell.
Introduction
GEMs are generated by combining
barcoded Gel Beads, transposed
nuclei, a Master Mix, and
Partitioning Oil on a Chromium
Chip E. To achieve single nuclei
resolution, the nuclei are delivered
at a limiting dilution, such that the
majority (~90-99%) of generated
GEMs contains no nuclei, while the
remainder largely contain a single
nucleus.
Upon GEM generation, the Gel
Bead is dissolved. Oligonucleotides
containing (i) an Illumina® P5
sequence, (ii) a 16 nt 10x Barcode
and (iii) a Read 1 (Read 1N)
sequence are released and mixed
with DNA fragments and Master
Mix. Thermal cycling of the GEMs
produces 10x barcoded single-
stranded DNA. After incubation,
the GEMs are broken and pooled
fractions are recovered.
Chromium Chip E
10x Barcoded
Gel Beads
GEMs
Nuclei,
Enzyme
Oil
Gel Beads
10x Barcoded DNA Fragments
Read 1N
10x
Barcode
Insert
Read 1N
10x
Barcode
Read 2N
P5
P5 Read 2N
Denaturation, Linear Amplification
Linear Amplification Product
Inside Individual GEMs
Read 1N
P5
10x
Barcode
Gel Beads
12Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
Step 4
Library Construction
Step 5
Sequencing
The Chromium Single Cell ATAC protocol produces Illumina®-ready sequencing
libraries. Illumina® sequencer compatibility, sample indices, sequencing depth & run
parameters, library loading and pooling are summarized.
Introduction
Step 3
Post GEM Incubation
Cleanup & QC
Silane magnetic beads are used to remove leftover biochemical reagents from the
post GEM reaction mixture. Solid Phase Reversible Immobilization (SPRI) beads are
used to eliminate unused barcodes from the sample.
P7, a sample index, and
Read 2 (Read2N) sequence
are added during library
construction via PCR.
The final libraries contain
the P5 and P7 primers
used in Illumina® bridge
amplification.
Chromium Single Cell ATAC Library
10x
Barcode
P5
Read 2N
Read 1N
P5
P5 Read 1N
10x
Barcode Read 2N P7
Pooled Amplified DNA Processed in Bulk
Sample
Index N
Priming
Sample Index PCR
Insert
Sample
Index N
P7
See Appendix for Oligonucleotide Sequences
P5 Read 1N
10x
Barcode Read 2N P7
Insert
Read 1N
Read 2N
i7:8
Sample
Index N
i5:16
14Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
Emulsion-safe
Plastics
Multiplet Rate
General
Reagent
Handling
50% Glycerol
Solution
• 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 ~1,000
1.6% ~3,075 ~2,000
2.3% ~4,625 ~3,000
3.1% ~6,150 ~4,000
3.9% ~7,700 ~5,000
4.6% ~9,250 ~6,000
5.4% ~10,750 ~7,000
6.2% ~12,300 ~8,000
6.9% ~13,850 ~9,000
7.7% ~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 move
reagents back to the recommended storage.
• Calculate reagent volumes with 10% excess of 1 reaction values.
• Cover Partitioning Oil tubes and reservoirs to minimize evaporation.
• Thoroughly mix samples with the beads during bead-based cleanup steps.
• 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.
Tips & Best Practices
Icons !
TIPS
Tips & Best Practices section
includes additional guidance
Signifies critical step requiring
accurate execution
Troubleshooting section includes
additional guidance
15Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
• Follow manufacturer’s calibration and maintenance schedules.
• Pipette accuracy is particularly important when using SPRIselect reagents.
• Minimize exposure of reagents, chips, and gaskets to sources of particles and fibers,
laboratory wipes, frequently opened flip-cap tubes, clothing that sheds fibers, and
dusty surfaces.
• 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 Recovery Wells.
• 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 aect the quality of the assay.
Chromium Chip
Handling
10x Chip
Holders
Chromium
Chip & Holder
Assembly
• 10x Chip Holders encase Chromium 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 side of
the holder together to unlock the lid and return
the holder to a flat position.
• Align notch on the chip (upper left
corner) and the holder.
• Insert the left-hand side of the
chip under the guide. Depress the
right-hand side of the chip until
the spring-loaded clip engages.
• Close the lid before dispensing
reagents into the wells.
Pipette
Calibration
Tips & Best Practices
10X Chip Holder
Guide
Clip
Sliders
Chromium Chip
10X Chip Holder
Assembled Chip
16Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
• Place the assembled chip and holder flat
on the bench with the lid closed.
• Dispense at the bottom of the wells without
introducing bubbles.
• Wait for the Cell Bead Mix to drain into the
bottom of the pipette tips and dispense
again to ensure complete volume transfer.
• Refer to Load Chromium Chip E for specific
instructions.
• Use one tube of Gel Beads per sample.
DONOT 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.
DONOT store Gel Beads at −20°C.
Chromium
ChipLoading
• Attach a 10x Vortex Adapter to the top of standard laboratory vortexers to vortex the
Gel Bead strips.
• After vortexing, remove the Gel Bead strip from the adapter. Flick the Gel Bead strip
in a sharp, downward motion to maximize Gel Bead recovery. Confirm there are no
bubbles at the bottom of the tubes.
• 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.
Gel Bead
Handling
Tips & Best Practices
• After reagents are loaded, attach the
gasket by holding the tongue (curved end,
to the right) and hook it 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. DO NOT press down on the top of
the gasket after attachment.
• Keep the assembly horizontal to avoid
wetting the gasket with Partitioning Oil.
10x Gasket
Attachment
Notched Cut
Tongue
17Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
• Oers two positions of the magnets (high
and low) relative to a tube, depending on
its orientation. Flip the magnetic separator
over to switch between high (magnet•High)
or low (magnet•Low) positions.
10x Magnetic
Separator
Tips & Best Practices
• 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 insucient mixing of sample and SPRIselect reagent will
lead to inconsistent results.
• Use fresh preparations of 80% Ethanol.
SPRIselect
Cleanup &
SizeSelection
Schematic 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 — 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 µl
Tutorial — Double Sided Size Selection
Step a – First SPRIselect: Add 50 µl SPRIselect reagent to 100 µl sample (0.5X).
Ratio
Step b – Second SPRIselect: Add 30 µl SPRIselect reagent to supernatant from step a (0.8X).
Ratio
= Volume of SPRIselect reagent added to the sample = 50 µl = 0.5X
Volume of DNA sample 100 µl
= Total Volume of SPRIselect reagent added to the sample (step a + b) = 50 µl + 30 µl = 0.8X
Original Volume of DNA sample 100 µl
Sample Indices in
Sample Index PCR
• Choose the appropriate sample index sets to ensure that no sample indices overlap in a
multiplexed sequencing run.
• Each well in the i7 Sample Index plate N contains a unique mix of 4 oligos.
• The sample indexes can therefore be used in any combination.
• Each sample index set is base-balanced to avoid monochromatic signal issues when it
is the sole sample loaded on an Illumina® sequencer.
19Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
1.0
Transposition GET STARTED!
Action Item 10x PN Preparation & Handling Storage
Equilibrateto
Room
Temperature
ATAC Buer 2000122 Vortex, centrifuge
briefly.
−20°C
Nuclei Buer*
*Concentrated 20X
stock; dilute 1:20 in
nuclease-free water
before use. (See below
to Prepare Diluted
Nuclei Buer)
2000153 Thaw. Vortex,
centrifuge briefly.
−20°C
Place on Ice ATAC Enzyme 2000123/
2000138
Centrifuge briefly. −20°C
Nuclei**
inDiluted Nuclei Buer
(See below to Prepare
Diluted Nuclei Buer)
**Refer to Demonstrated Protocol Nuclei Isolation for ATAC Sequencing
(Document CG000167) for isolating nuclei. Adhering to this protocol is critical
for optimal assay performance. If following a dierent nuclei isolation protocol,
use the Diluted Nuclei Buer for final nuclei pellet suspension.
The use of the Tris-based Diluted Nuclei Buer for nuclei suspension is critical
for optimal assay performance. The composition of the Diluted Nuclei Buer,
including Magnesium concentration, has been optimized for the Transposition
and Barcoding steps. Suspension of nuclei in a dierent buer may not be
compatible with the downstream protocol steps.
Prepare Diluted Nuclei
Buer
!
!
Diluted Nuclei Buer
Maintain at 4°C
Stock Final 1 ml
Nuclei Buer
(PN-2000153)
Nuclease-free Water
20X
-
1X
-
50 µl
950 µl
Step 1 Transposition
20Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
Step 1 Transposition
Based on the Targeted Nuclei Recovery, resuspend the nuclei in Diluted Nuclei Buer to get
corresponding 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)
500 155-390
1,000 310-780
2,000 610-1,540
3,000 925-2,300
4,000 1,230-3,075
5,000 1,540-3,850
6,000 1,850-4,600
7,000 2,150-5,400
8,000 2,460-6,150
9,000 2,770-6,900
10,000 3,080-7,700
Calculate volume of Nuclei Stock and Diluted Nuclei Buer for a total volume of 5 µl
Volume of Nuclei Stock (µl) =
Volume of Diluted Nuclei Buer* (µl) = 5 µl - volume of Nuclei Stock (µl)
*Use ONLY Diluted Nuclei Buer (Dilute Nuclei Buer (PN-2000153) 1:20 in nuclease-free water)
Targeted Nuclei Recovery x 1.53 (Recovery eciency factor)
Nuclei Stock Concentration (nuclei/ µl)
Example Calculation
Targeted Nuclei Recovery = 4000 nuclei
Nuclei Stock Concentration = 2500 nuclei/ µl
Recovery eciency factor 1.53
Volume of Nuclei Stock (µl) =
Volume of Diluted Nuclei Buer = 5 µl - 2.45 ul = 2.55 µl
Add calculated volumes of Diluted Nuclei Buer and Nuclei Stock to the Transposition Mix in
step 1.1
Targeted Nuclei Recovery x 1.53 (Recovery eciency factor) = 4000 x 1.53 = 2.45 µl
Nuclei Stock Concentration (nuclei/µl) 2500
Nuclei Concentration
Guidelines
21Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
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 Buer 2000122 7.0 30.8 61.6
ATAC Enzyme 2000123/
2000138 3.0 13.2 26.4
Total -10.0 44.0 88.0
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 and
Diluted Nuclei Buer for a total volume of 5 µl.
d. Add the calculated volume of Diluted Nuclei Buer to the Transposition Mix. Pipette
mix. Centrifuge briefly.
e. 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
50°C 15 µl 60 min
Step Temperature Time
Incubate 37°C 00:60:00
Hold 4°C Hold
!
1.1
Prepare
TranspositionMix
Step 1 Transposition
1.2
Isothermal Incubation
23Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
2.0
GEM Generation &
Barcoding
Step 2 GEM Generation & Barcoding
GET STARTED!
Action Item 10x PN Preparation & Handling Storage
Equilibrateto
Room
Temperature
Chromium
SingleCell ATAC
Gel Beads
2000132 Equilibrate to room
temperature 30 min
before loading the chip.
−80°C
Nuclease-free
Water
- - -
Reducing Agent B 2000087 Thaw, vortex, verify no
precipitate, centrifuge
briefly.
−20°C
Barcoding Reagent 2000124 Thaw, vortex, verify no
precipitate, centrifuge
briefly.
−20°C
Place on Ice Barcoding Enzyme 2000125/
2000139
Maintain on ice. Store at
−20°C immediately after
use.
−20°C
Obtain Partitioning Oil 220088 -Ambient
Chromium Chip E 2000121 See Tips & Best Practices. Ambient
10x Gasket 370017/
3000072
See Tips & Best Practices. Ambient
10x Vortex Adapter 330002 See Tips & Best Practices. Ambient
10x Chip Holder 330019 See Tips & Best Practices Ambient
50% glycerol
solution
If using <8 reactions
-See Tips & Best Practices. -
Firmware Version 3.16 or higher
is required in the Chromium
Controller or the Single Cell
Chromium Controller used for the
Single Cell ATAC protocol.
!
24Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
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 2000124 61.5 270.6 541.2
Reducing Agent B 2000087 1.5 6.6 13.2
Barcoding Enzyme 2000125/
2000139 2.0 8.8 17.6
Total -65.0 286.0 572.0
2.1
Prepare Master Mix
Step 2 GEM Generation & Barcoding
25Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
a. Assemble Chromium Chip E in a 10x Chip Holder See Tips & Best Practices
b. Dispense 50% Glycerol Solution into Unused Chip Wells (if <8 samples per chip)
i. 75 µl into unused wells in row labeled 1.
ii. 40 µl into unused wells in row labeled 2.
iii. 240 µl into unused wells in row labeled 3.
c. Prepare Master Mix + Transposed Nuclei
Add 65 µl Master Mix to each tube containing Transposed Nuclei for a total of 80 µl in each
tube.
d. Load Row Labeled 1
Gently pipette mix the Master Mix +
Transposed Nuclei. Using the same
pipette tip, dispense 75 µl Master Mix +
Transposed Nuclei into the bottom center
of each well in row labeled1 without
introducing bubbles. Wait 30 sec.
If volume is <75 µl, load available volume,
which should not be <70 µl.
e. Prepare Gel Beads
Snap the Gel Bead strip into a 10x Vortex Adapter. Vortex 30 sec. Remove the Gel Bead
strip and flick in a sharp, downward motion to ensure maximum recovery. Confirm there
are no bubbles at the bottom of the tubes and liquid levels look even.
f. Load Gel Beads in Row Labeled 2
Puncture the foil seal of the Gel Bead
tubes. Slowly aspirate 40 µl Gel Beads.
Dispense into the bottom of each well in
row labeled2 without introducing bubbles.
g. Load Row Labeled 3
Dispense 240 µl Partitioning Oil into each
well in row labeled 3.
Failure to add Partitioning Oil can damage
the Chromium Controller.
h. Attach 10x Gasket
Align the notch with the top left-hand
corner. Ensure the gasket holes are
aligned with the wells. Avoid touching the
smooth gasket surface. DONOT press
down on the gasket.
TIPS
75 µl Master Mix +
Transposed Nuclei
40 µl Gel Beads
240 µl Partitioning Oil
!
Keep horizontal to
avoid wetting the
gasket.
See Tips & Best Practices for chip handling instructions. When loading the chip, raising
and depressing the pipette plunger should each take ~5 sec. When dispensing, raise the
pipette tips at the same rate as the liquid is rising, keeping the tips slightly submerged.
2.2
Load Chromium Chip E
DO NOT add 50% glycerol solution to the
top row of Recovery Wells. DO NOT use any
substitute for 50% glycerol solution.
Step 2 GEM Generation & Barcoding
26Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
a. Press the eject button on the Controller to
eject the tray.
b. Place the assembled chip with the gasket in
the tray. Press the button to retract the tray.
c. Confirm the program on screen. Press the
play button.
d. At completion of the run (~7 min), the
Controller will chime. Immediately proceed
to the next step.
a. Place a PCR 8-tube strip on ice.
b. Press the eject button of the Controller to
remove the chip.
c. Discard the gasket. Open the chip holder.
Fold the lid back until it clicks to expose the
wells at 45 degrees.
d. Check the volume in rows 1-3. Abnormally
high volume in any well indicates a clog.
e. Slowly aspirate 100 µl GEMs from the lowest
points of the Recovery Wells without creating
a seal between the pipette tips and the wells.
f. Withdraw pipette tips from the wells. GEMs
should appear opaque and uniform across
all channels. Excess Partitioning Oil (clear) in
the pipette tips indicates a potential clog.
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.
!
2.3
Run the Chromium
Controller
2.4
Transfer GEMs
Step 2 GEM Generation & Barcoding
Expose Wells at 45 Degrees
Transfer GEMs
GEMs
27Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
Use a thermal cycler that can accommodate at least 100 µl volume. A volume of
125µl is the preferred setting on Bio-Rad C1000 Touch. In alternate thermal cyclers,
use highest reaction volume setting.
a. Incubate in a thermal cycler with the following protocol.
Lid Temperature Reaction Volume Run Time
105°C 125 µl 30 min
Step Temperature Time
172°C 00:05:00
298°C 00:00:30
398°C 00:00:10
459°C 00:00:30
572°C 00:01:00
Go to step 3, repeat 11X
(Total 12 cycles)
615°C Hold
2.5
GEM Incubation
Step 2 GEM Generation & Barcoding
29Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
3.0
Post GEM Incubation
Cleanup & QC
GET STARTED!
Action Item 10x PN Preparation & Handling Storage
Equilibrateto
Room
Temperature
Reducing Agent B 2000087 Thaw, vortex, verify no
precipitate, centrifuge
briefly.
−20°C
Nuclease-free
Water - - -
Dynabeads MyOne
SILANE
2000048 Vortex thoroughly
(≥30sec) to resuspend
beads immediately before
use.
4°C
Beckman Coulter
SPRIselect
Reagent
-Manufacturer’s
recommendations.
-
Thaw at 65°C Cleanup Buer 2000088 Thaw for 10 min at
65°C at max speed on a
thermomixer. Verify there
are no visible crystals.
Cool to room temperature.
−20°C
Obtain Recovery Agent 220016 -Ambient
Qiagen Buer EB -Manufacturer’s
recommendations.
-
Bio-Rad 10%
Tween 20
-Manufacturer’s
recommendations.
-
10x Magnetic
Separator
230003 -Ambient
Prepare
80%Ethanol
Prepare 10 ml for
8 reactions
-Prepare fresh. -
Step 3 Post GEM Incubation Cleanup & QC
30Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
a. 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.
The resulting biphasic mixture contains
Recovery Agent/Partitioning Oil (pink) and
aqueous phase (clear), with no persisting
emulsion (opaque).
A smaller aqueous phase volume indicates a
clog during GEM generation.
b. Slowly remove 125 µl Recovery Agent/
Partitioning Oil (pink) from the bottom of the
tube. DO NOT aspirate any aqueous sample.
c. Prepare Dynabeads Cleanup Mix.
!
3.1
Post GEM Incubation
Cleanup – Dynabeads
Dynabeads Cleanup Mix
Add reagents in the order listed PN 1X (µl) 4X +
10% (µl)
8X +
10% (µl)
Cleanup Buer 2000088 182 800.8 1601.6
Dynabeads MyOne SILANE
Vortex thoroughly (≥30 sec) immediately
before adding to the mix.
2000048 835.2 70.4
Reducing Agent B 2000087 522 44
Nuclease-free Water -522 44
Total -200 880 1760
d. Vortex and add 200 µl to each sample.
Pipette mix 5x (pipette set to 200 µl).
e. Incubate 10 min at room temperature.
Step 3
Biphasic Mixture
Remove Recovery Agent
Add Dynabeads Cleanup Mix
Post GEM Incubation Cleanup & QC
31Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
f. Prepare Elution Solution I. Vortex and centrifuge briefly.
Elution Solution I
Add reagents in the order listed PN 1X (µl) 4X +
10% (µl)
8X +
10% (µl)
Buer EB -98.0 431.2 862.4
10% Tween 20 -1.0 4.4 8.8
Reducing Agent B 200087 1.0 4.4 8.8
Total -100.0 440.0 880.0
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
30sec.
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 40.5 µl Elution Solution I 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 40 µl sample to a new tube strip.
TIPS
Step 3 Post GEM Incubation Cleanup & QC
32Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
3.2
Post GEM Incubation
Cleanup – SPRIselect
Step 3 Post GEM Incubation Cleanup & QC
a. Vortex the SPRIselect reagent until fully resuspended. Add 48 µl SPRIselect reagent
to each sample. Pipette mix thoroughly.
b. 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.
j. Remove the tube strip from the magnet. Immediately add 40.5 µl Buer EB.
k. Pipette mix (pipette set to 30 µl) without introducing bubbles.
l. Incubate 2 min at room temperature.
m. Centrifuge briefly. Place on the magnet•Low until the solution clears.
n. Transfer 40 µl sample to a new tube strip.
o. Store at 4°C for up to 72 h or at −20°C for up to 2 weeks, or proceed to the next step.
STOP
34Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
4.0
Library Construction
GET STARTED!
Action Item 10x PN Preparation & Handling Storage
Equilibrateto
Room
Temperature
Chromium i7
Sample Index
Plate N, Set A
3000262 -−20°C
Beckman Coulter
SPRIselect
Reagent
-Manufacturer’s
recommendations.
-
Agilent
Bioanalyzer DNA
kit
(if used for QC)
-Manufacturer’s
recommendations.
-
Place on Ice 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.
-
Obtain Qiagen Buer EB --Ambient
10x Magnetic
Separator
230003 See Tips & Best Practices. Ambient
Prepare
80% Ethanol
Prepare 10 ml
for 8 reactions
-Prepare fresh. Ambient
Step 4 Library Construction
35Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
Choose the appropriate sample index sets to ensure that no sample indices
overlap 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
Total -57.5 253 506
b. Add 57.5 µl Sample Index PCR Mix to 40 µl sample. Pipette mix and centrifuge briefly.
c. Add 2.5 µl of an individual Chromium i7 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
105°C 100 µl ~30 min
Step Temperature Time
198°C 00:00:45
298°C 00:00:20
367°C 00:00:30
472°C 00:00:20
Go to step 2, see table below
for # cycles
572°C 00:01:00
64°C Hold
Cycle Number Optimization Table
Targeted Nuclei
Recovery
Cell Lines
Total Cycles
Primary Cells
Total Cycles
500-2,000 12 13
2,001-6,000 11 12
6,001-10,000 10 11
e. Store at 4°C for up to 72 h or proceed to the next step.
STOP
4.1
Sample Index PCR
Step 4 Library Construction
The table recommends a starting point
for cycle number optimization for
cell lines and primary cells based on
Targeted Nuclei Recovery.
36Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
4.2
Post Sample Index
Double Sided Size
Selection – SPRIselect
a. Vortex to resuspend SPRIselect reagent. Add 40 µl SPRIselect reagent to each
sample. Pipette mix.
b. Incubate 5 min at room temperature.
c. Place on the magnet•High until the solution clears.
d. Transfer 130 µl supernatant to a new strip tube. DO NOT discard the supernatant.
e. Vortex to resuspend SPRIselect reagent. Add 74 µl SPRIselect reagent to each
sample. 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 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.
m. Remove remaining ethanol.
n. Remove from the magnet. Immediately add 20.5 µl Buer EB. Pipette mix.
o. Incubate 2 min at room temperature.
p. Centrifuge briefly. Place on the magnet•Low until the solution clears.
q. Transfer 20 µl sample to a new tube strip.
r. Store at 4°C for up to 72 h or at −20°C for long-term storage.
TIPS
STOP
Step 4 Library Construction
37Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
a. EITHER Run 1 µl sample on the Agilent Bioanalyzer High Sensitivity DNA chip to
determine fragment size.
b. OR Run 2 µl sample on the Agilent TapeStation D1000 ScreenTape to determine
fragment size.
Representative Trace
4.3
Post Library Construction
QC
Step 4 Library Construction
Representative Trace
38Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
a. Thaw KAPA Library Quantification Kit for Illumina® Platforms.
b. Dilute 1 µl sample with deionized water to appropriate dilutions that fall within the
linear 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
195°C 00:03:00
295°C 00:00:05
367°C 00:00:30
4Go 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 using the
average size in the region of 150-1000 bp.
4.4
Post Library Construction
Quantification
Step 4 Library Construction
For Library Construction related questions, contact support@10xgenomics.com
40Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
Chromium Single Cell ATAC libraries comprise double stranded DNA fragments
which begin with P5 and end with P7. Sequencing these libraries produces a standard
Illumina® BCL data output folder.
The BCL data for Single Cell ATAC libraries include:
•Paired-end Read 1N containing insert sequence only
•Read 2N containing insert sequence, starting from the opposite end of fragment
•8 bp sample index in the i7 read
•16 bp 10X barcode sequence in the i5 read
The Cell Ranger scATAC pipeline performs demultiplexing and leverages the 10x
Barcodes to group read-pairs and associate them to individual cells for secondary
analysis and visualization. In addition to performing standard analysis steps such as
alignment, Cell Ranger scATAC leverages the 10x Barcodes to generate chromatin
accessibility data with single cell resolution. This enables applications including cell
clustering, cell type classification, and dierential accessibility at a scale of hundreds
to thousands of cells.
The compatibility of the listed sequencers has been verified by 10x Genomics. Some
variation 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)
•HiSeq 2500™ (Rapid Run)
•HiSeq™ 3000/4000
•NovaSeq™
Each sample index in the Chromium i7 Sample Index Plate Kit N, Set A (PN-3000262)
is a mix of 4 dierent sequences to balance across all 4 nucleotides. If multiple
samples are pooled in a sequence lane, the sample index name (i.e. the Chromium
i7 Sample Index Plate N, Set A well ID) is needed in the sample sheet used for
generating FASTQs with “cellranger-dna mkfastq”.
Illumina® Sequencer
Compatibility
Sample Indices
Sequencing Depth & Run
Parameters
Sequencing
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 Recommended Number of Cycles
Read 1N
i7 Index
i5 Index
Read 2N
50 cycles
8 cycles
16 cycles
50 cycles
Sequencing Libraries
Step 5
P5 Read 1N
10x
Barcode Read 2N P7
Insert
Read 1N
Read 2N
i7:8
Sample
Index N
i5:16 bp
41Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
Sequencing
Once quantified and normalized, Single Cell ATAC libraries should be denatured and
diluted according to the table below. Consult the Technical Note on Sequencing Metrics
and Base Composition of Chromium Single Cell ATAC Libraries (DocumentCG000181),
available at the 10x Genomics Support website, for more information.
Instrument Loading Concentration (pM) PhiX (%)
MiSeq™11 1
NextSeq™ 500 1.7 1
HiSeq™ 2500 (RR) 11 1
HiSeq™ 4000 180 1
NovaSeq™250 1
Pooling dissimilar libraries may compromise the ability to pool eectively due
to dierences in insert sizes. DO NOT pool Single Cell ATAC libraries with other
10xGenomics libraries.
Library Loading
Step 6
Library Pooling
43Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
STEP NORMAL REAGENT CLOGS & WETTING FAILURES
2.4 d
After Chip E is
removed from the
Controller and the
wells are exposed
All 8 Recovery Wells are similar in
volume and opacity.
Recovery Well G indicates a reagent clog.
Recovery Well C and E indicate a wetting
failure. Recovery Wells B, D, and F are
normal. Wells A and H contain 50% Glycerol
Solution.
The image indicates clogs in the Gel Bead
line (orange arrow) and the sample line
(yellow arrow) as evidenced by higher than
usual volumes in the input wells.
2.4 e
Transfer GEMs from
Chip E Recovery
Wells
All liquid levels are similar in volume and
opacity without air trapped in the pipette
tips.
Pipette tips C and E indicate a wetting
failure. Pipette tip C contains partially
emulsified GEMs. Emulsion is absent
in pipette tip E. Pipette tip G indicates a
reagent clog.
A B C D E F G H A B C D E F G H
6.1 GEMs
Troubleshooting
A B C D E F G H
44Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
STEP NORMAL REAGENT CLOGS & WETTING FAILURES
3.1 a
After transfer of the
GEMs +
Recovery Agent
All liquid levels are similar in the
aqueous sample volume (clear) and
Recovery Agent/Partitioning Oil (pink).
Tube G indicates a reagent clog has
occurred. There is a decreased volume of
aqueous layer (clear).
Tube C and E indicate a wetting failure has
occurred. There is an abnormal residual
volume of Recovery Agent/Partitioning Oil
(pink).
3.1 b
After aspiration of
Recovery Agent/
Partitioning Oil
All liquid volumes are similar in the
aqueous sample volume (clear) and
residual Recovery Agent/Partitioning Oil
(pink).
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
All liquid volumes are similar after
addition of the Dynabeads Cleanup Mix.
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).
Troubleshooting
If a channel clogs or wetting failure occurs during GEM generation, it is
recommended 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.
A B C D E F G H A B C D E F G H
A B C D E F G H A B C D E F G H
A B C D E F G H
A B C D E F G H
45Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
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 10x Chip
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 there is a 10x
Gasket on the Chromium Chip. In the case when the 10x Gasket installation was
forgotten, install and try again. In the case when a 10x Gasket was already installed,
remove, reapply, and try again. If the error message is still received after trying either
of these more than twice, contact support@10xgenomics.com for further assistance.
c. Pressure not at Setpoint:
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 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 Chip
must be discarded. Do not try running this Chromium Chip again as this may
damage the Chromium Controller.
d. CAUTION: Chip Holder not Present: Eject the tray by pressing the eject button to
check there is a 10x Chip Holder encasing the Chromium Chip. In the case when the
10x Chip Holder was forgotten, install with a 10x Gasket in place, and try again. If
the error message is still received after a 10x Chip Holder is confirmed as in place,
contact support@10xgenomics.com for further assistance.
e. Invalid Chip CRC Value: This indicates the Chromium Chip has encountered an error,
should not be run, and must be discarded. Contact support@10xgenomics.com for
further assistance.
6.2
Chromium Controller
Errors
Troubleshooting
47Click to TOC Chromium Single Cell ATAC Reagent Kits User Guide | Rev A
Oligonucleotide
Protocol steps correspond to the Chromium Single Cell ATAC Reagent Kits User Guide (CG000168)
Protocol Step 1 – Transposition
Transposition Mix Read 1N primer sequence:
5ʹ-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3ʹ
Read 2N primer sequence:
5ʹ-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3ʹ
Transposed DNA
Product
5ʹ-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-----insert-- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC-3ʹ
3ʹ-AGCAGCCGTCGCAGTCTACACATATTCTCTGTC --insert-----GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG-5ʹ
Protocol Step 2.5 – GEM Incubation
Gel Bead Oligo
Primer
PN-2000132
5’-AATGATACGGCGACCACCGAGATCTACAC-NNNNNNNNNNNNNNNN-TCGTCGGCAGCGTC-3’
Linear Amplification
DNA Product
5ʹ-AATGATACGGCGACCACCGAGATCTACAC-NNNNNNNNNNNNNNNN-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG----insert----CTGTCTCTTATACACATCTCCGAGCCCACGAGAC-3ʹ
Protocol Step 4.1 – Sample Index PCR
SI-PCR Primer B
PN-2000128
i7 Sample Index
PlateN, Set A
PN-3000262
Forward Primer:
5’-AATGATACGGCGACCACCGAGA-3’
Reverse Primer:
5’- CAAGCAGAAGACGGCATACGAGAT-NNNNNNNN-GTCTCGTGGGCTCGG-3ʹ
Sample Index PCR
Product
5ʹ-AATGATACGGCGACCACCGAGATCTACAC-NNNNNNNNNNNNNNNN-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG---insert---CTGTCTCTTATACACATCTCCGAGCCCACGAGAC-NNNNNNNN-ATCTCGTATGCCGTCTTCTGCTTG-3ʹ
3ʹ-TTACTATGCCGCTGGTGGCTCTAGATGTG-NNNNNNNNNNNNNNNN-AGCAGCCGTCGCAGTCTACACATATTCTCTGTC---insert---GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG-NNNNNNNN-TAGAGCATACGGCAGAAGACGAAC-5ʹ
Read 1N Read 2N
Gel
Bead P5 10x
Barcode Partial
Read 1N
Partial P5 Partial
Read 2N
Sample
Index N
P7
Read 2N P7
Sample
Index N
P5 10x
Barcode Read 1N Insert
Appendix
Sequences
P5 10x
Barcode Read 1N Read 2N
Insert
