Nov 28, 2025

Public workspaceLow Cell Input Nuclei Isolation for Single Cell ATAC-Seq

Forked from a private protocol
DOI
dx.doi.org/10.17504/protocols.io.bp2l6n1xkgqe/v1
  • Carmen Sancho1
  • 1Wellcome Sanger Institute
  • Vento-Tormo
    Tech. support email: rv4@sanger.ac.uk
Regina Hoo
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DOI: https://dx.doi.org/10.17504/protocols.io.bp2l6n1xkgqe/v1
Protocol Citation: Carmen Sancho 2025. Low Cell Input Nuclei Isolation for Single Cell ATAC-Seq. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6n1xkgqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: November 22, 2019
Last Modified: November 28, 2025
Protocol Integer ID: 30096
Keywords: ATAC-seq, single cell atac sequencing, single cell atac, low cell input nuclei isolation, methodology of nuclei isolation, nuclei isolation, seq this protocol, seq
Disclaimer
Handling, use, storage, and disposal of human tissues must be conducted in a respectful manner in line with Human Tissue Authority (HTA) guidelines, reflecting the sensitive nature and origin of the material.
Ensure prior to start that collection, carriage, and receipt of tissues is compliant with all HTA guidelines, including Research Ethics Service/Committee approval, and thorough labelling and tracking of all human materials.
Abstract
This protocol describes the the methodology of nuclei isolation for Single Cell ATAC Sequencing adopted from 10X Genomics guide CG000169 Revision D.
Materials
MATERIALS
ReagentBSAMerck MilliporeSigma (Sigma-Aldrich)Catalog #A7906
ReagentFBSInvitrogen - Thermo Fisher
ReagentFlowmi™ Cell Strainer 40 μm Bel-ArtCatalog #H13680-0040
ReagentPBS
ReagentTween-20
ReagentRPMI 1640 MediumThermo Fisher ScientificCatalog #11875093
ReagentSingle Cell ATAC Library and Gel Bead Kit10x GenomicsCatalog #PN-1000175
ReagentDigitonin (5%)Thermo Fisher ScientificCatalog #BN2006
ReagentTrizma® hydrochloride solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #T2194-1L
ReagentNonidet™ P 40 SubstituteMerck MilliporeSigma (Sigma-Aldrich)Catalog #74385
ReagentSodium chloride solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #59222C
ReagentMACS BSA Stock SolutionMiltenyi BiotecCatalog #130-091-376
ReagentMagnesium chloride solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #M1028
Troubleshooting
Prepare diluted nuclei buffer

Keep nuclei buffer on ice!
ProductStockFinalVolume (1 ml)
Nuclei buffer (20x) PN-2000153/200020720X1x50 ul
Nuclease-free water950 ul
**location at:

Prepare buffer A
Keep buffer A on ice and prepare fresh!
ProductStockFinalVolume (4.94 ml)
Tris-HCl (pH 7.4)1M10 mM50 ul
NaCl5M10 mM10 ul
MgCl21M3 mM15 ul
BSA10%1%500 ul
Tween-2010%0.1%50 ul
Nuclease-free water4.315 ml

Prepare wash buffer
Keep wash buffer on ice and prepare fresh!
ProductStockFinalVolume (2 ml)
Buffer A1.976 ml
Nuclease-free water24 ul
**incubate Digotonin at 65 degree celcius to dissolve precipitate before use.

Prepare lysis buffer
Keep lysis buffer on ice and prepare fresh!
ProductStockFinalVolume (2 ml)
Buffer A1.976 ml
Nonidet P40 Substitute (if using Sigma (74385) 100% solution, prepare 10% stock10%0.1%20 ul
Digitonin5%0.01%4 ul

Additional buffers
PBS + 0.04% BSA (maintain at Temperature4 °C )

ProductStockVolume
PBS5 ml
BSA (0.04%)10%20 ul

Nuclei isolation from fresh cells
Nuclei may be isolated from 2000-100,000 cells using this protocol. Cells are already resuspended in PBS + 0.04% BSA.

Centrifuge cell suspension at 300 rcf for 5min at Temperature4 °C and re-suspend the cell pellet in 50 ul of PBS 0.04% BSA. Transfer 50 ul cell suspension to a 0.2ml tube. Proceed to next step.

Centrifuge at 300 rcf for Duration00:05:00 at Temperature4 °C .


Remove 45ul supernatant without touching the bottom of the tube to avoid dislodging the cell pellet.
Add Amount45 µL chilled Lysis Buffer. Gently pipette mix 3x.

Incubate for 4 min* on ice.

*Time between 3-5min, depending on cell type.
Add Amount50 µL chilled Wash Buffer to the tube. DO NOT MIX!

Centrifuge at 500 rcf for Duration00:05:00 at Temperature4 °C .


Remove 95ul supernatant without disrupting the nuclei pellet.
Add Amount45 µL chilled Diluted Nuclei Buffer to the pellet. DO NOT MIX.

Centrifuge at 500 rcf for Duration00:05:00 at Temperature4 °C .
Remove the supernatant in 2 step without touching the bottom of the tube to avoid dislodging the nuclei pellet.


Resuspend the nuclei pellet in Amount5.5 µL chilled diluted nuclei buffer (pellet may not be visible).

**If original cell count <20,000 cells, do not need to count nuclei. Load everything into 10X.


Use Amount2.5 µL nuclei suspension mix with Amount2.5 µL Diluted Nuclei Buffer and Amount5 µL Trypan blue to determine cell count.


nuclei count= _____ x 2 x 2 x 10 nuclei/ul




Proceed to load about Amount5 µL of nuclei suspension for transposition step.

Prepare transposition mix



GEMS generation
Proceed immediately to Chromium Single Cell ATAC Solution User Guide PAGE 24

CG000168_ChromiumSingleCell_ATAC_ReagentKits_UserGuide_RevC.pdf