Scale-scATAC

Koen Theunis

Jan 14, 2026

Scale-scATAC

DOI

https://dx.doi.org/10.17504/protocols.io.4r3l2z24ql1y/v1

Koen Theunis^1,2,3,4

¹Laboratory of Computational Biology, VIB Center for AI & Computational Biology, Leuven, Belgium;
²VIB-KU Leuven Center for Brain & Disease Research, Leuven, Belgium;
³Department of Human Genetics, KU Leuven, Leuven, Belgium;
⁴Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, United States

Aertslab

Koen Theunis

Laboratory of Computational Biology, VIB Center for AI & Com...

DOI: https://dx.doi.org/10.17504/protocols.io.4r3l2z24ql1y/v1

Protocol Citation: Koen Theunis 2026. Scale-scATAC. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2z24ql1y/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

We use this protocol and it's working

Created: January 14, 2026

Last Modified: January 14, 2026

Protocol Integer ID: 238543

Keywords: ASAPCRN, scATAC-seq, scaleATAC, based snatac, scatac this protocol, scatac, droplet, seq, scale, barcoded tn5

Funders Acknowledgements:

Aligning Science Across Parkinson’s

Grant ID: ASAP-000430

Aligning Science Across Parkinson’s

Grant ID: ASAP-025179

Abstract

This protocol allows you to scale your droplet based snATAC-seq by pre-indexing with barcoded Tn5. 

Attachments

Scale-scATAC.xlsx

19KB

Materials

 
20X
  Diluted Nuclei BufferStockFinalVolume
Tris-HCl pH 7.41000mM200mM200µl
MgCl₂1000mM100mM100µl
Nuclease-free Water  700µl
Total Volume    1000µl
  
Diluted
  Nuclei BufferStock FinalVolume
20X Nuclei Buffer (10x
  Genomics)20X1X50µl
Nuclease-free Water  950µl
Total Volume    1000µl
  
Diluted
  Nuclei BufferStock FinalVolume
Tris-HCl pH 7.41000mM10mM10µl
MgCl₂1000mM5mM5µl
Nuclease-free Water  985µl
Total Volume    1000µl
  
Tagmentation
  Buffer V1StockFinalVolume
Tris-HCl pH 7.41000mM10mM0,1µl
DMF100%15%1,5µl
MgCl₂1000mM5mM0,05µl
Nuclease-free Water    5,35µl
Total Volume    7µl
  
Tagmentation
  BufferStock Final (in 15uL)Volume
Tris-HCl pH 7.4 (or Tris
  acetate 7.6)1000mM10mM0,10µl
DMF100%10%1,50µl
Pitstop1000uM70uM1,05 
MgCl₂ (or Mg acetate)1000mM5mM0,05µl
Nuclease-free Water  4,30µl
Total Volume    7µl
  
Loading
  buffer w/o SBSStock conc  Final conc V(µl)
Tris-HCl pH 7.41000mM10mM5
DMF100%10%50
MgCl21000mM5mM2,5
NaCl5000mM20mM2
Glycerol (filtered)50%10%100
Nuclease-free Water  340,5
Total Volume    500
  
Final
  Loading bufferStock conc  Final conc V(µl)
Loading buff w/o SBS  95
short SBS oligo100uM5uM5
Total Volume    100
  
NameOligo for loading buffer
Short SBSCGTGTGCTCTTCCGATCT
  
Namei7 index primerReverse complement index
s3_i7_S701CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTAAGGCGA
s3_i7_S702CAAGCAGAAGACGGCATACGAGATCTAGTACGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCGTACTAG
s3_i7_S703CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAGGCAGAA
s3_i7_S706CAAGCAGAAGACGGCATACGAGATCATGCCTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTAGGCATG
s3_i7_S707CAAGCAGAAGACGGCATACGAGATGTAGAGAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTCTCTAC
s3_i7_S708CAAGCAGAAGACGGCATACGAGATCCTCTCTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCAGAGAGG
s3_i7_T267CAAGCAGAAGACGGCATACGAGATTACTCTTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAAGAGTA
s3_i7_T268CAAGCAGAAGACGGCATACGAGATAACTAACCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGTTAGTT
s3_i7_T269CAAGCAGAAGACGGCATACGAGATTCCTCAATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATTGAGGA
s3_i7_T271CAAGCAGAAGACGGCATACGAGATGCTGCTTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAAGCAGC
s3_i7_T272CAAGCAGAAGACGGCATACGAGATACCGAATCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGATTCGGT
s3_i7_T273CAAGCAGAAGACGGCATACGAGATCTCCAGAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTCTGGAG
s3_i7_T274CAAGCAGAAGACGGCATACGAGATTGCTAGTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGACTAGCA
s3_i7_T275CAAGCAGAAGACGGCATACGAGATCGACCTGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGCAGGTCG
s3_i7_T276CAAGCAGAAGACGGCATACGAGATAGCCAGCCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGCTGGCT
s3_i7_T278CAAGCAGAAGACGGCATACGAGATGGTATGGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCCATACC
 

Troubleshooting

Before start

Prepare barcoded Tn5 with: dx.doi.org/10.17504/protocols.io.j8nlk1w1wg5r/v1

Prepare nuclei

 Count and dilute nuclei

 

Resuspend nuclei in 1X DNB

Count nuclei

Dilute nuclei to desired concentration in 1X DNB
 
ABC
Number
  of Nuclei per Well Required Nuclei Concentration
  (Nuclei/μl) Approx. Final Output of
  Well 
20,000 4,000 5,000 
25,000 5,000 6,250 
30,000 6,000 7,500 
35,000 7,000 8,750 
40,000 8,000 10,000 
45,000 9,000 11,250 
50,000 10,000 12,500 
 

Tagmentation

1h 6m

Mix diluted nuclei with tagmentation buffer

For 24 wells: mix 150 µL diluted nuclei with 210 µL Tagmentation Buffer

Add 12 uL nuclei/TB-mix to each well while keeping plate on ice.

Use a multichannel-channel pipet set to 12 µL to mix nuclei 5 times (no bubbles please)

Seal plate. DO NOT SPIN PLATE. Immediately proceed to tagmentation

Tagmentation

Tagment for Duration01:00:00   @Temperature37 °C   in a thermocycler block with heated lid (47°C)

Stop tagmentation

Take plate from thermocycler and place Duration00:05:00   on ice.

Take multi-channel pipet, pipet gently 3x, and combine samples to same row.

Pool all wells in 1.5mL DNA LoBind tube

Centrifuge Centrifigation400 x g, 4°C, 00:06:00  

Remove supernatant without disturbing nuclei pellet

Add 30 uL of loading buffer

Count nuclei

Prepare for HyDrop-ATAC or 10X ATAC

Prepare PCR buffer for HyDrop-ATAC or Barcoding reagent for 10x Genomics ATAC.

Dilute nuclei to desired concentration with loading buffer and take 15 µL

Add PCR mix

Run sample on HyDrop or 10x Genomics

PCR

First PCR: limit cycles to 4

Second (index) PCR: use 2.5 µL of the i7 index primer (see Oligo's) instead of the 10x Index primers. P5 can be used from the kit.
 #Cycles: 8 to 10

Sequencing
 
ABCD
R1R2i7i5
4074816
Depending on the sequencing kit, R1 and R2 can be extended.
 

Protocol references

Mulqueen, R.M., Pokholok, D., O’Connell, B.L. et al. High-content single-cell combinatorial indexing. Nat Biotechnol 39, 1574–1580 (2021). https://doi.org/10.1038/s41587-021-00962-z

https://scale.bio/wp-content/uploads/2023/05/ScaleBio_scATAC_pre-indexing_protocol_v1.6_24Nov22.pdf


20X Diluted Nuclei Buffer	Stock	Final	Volume
Tris-HCl pH 7.4	1000	mM	200	mM	200	µl
MgCl₂	1000	mM	100	mM	100	µl
Nuclease-free Water					700	µl
Total Volume					1000	µl


Diluted Nuclei Buffer	Stock	Final	Volume
20X Nuclei Buffer (10x Genomics)	20	X	1	X	50	µl
Nuclease-free Water					950	µl
Total Volume					1000	µl


Tagmentation Buffer V1	Stock	Final	Volume
Tris-HCl pH 7.4	1000	mM	10	mM	0,1	µl
DMF	100	%	15	%	1,5	µl
MgCl₂	1000	mM	5	mM	0,05	µl
Nuclease-free Water					5,35	µl
Total Volume					7	µl


Tagmentation Buffer	Stock	Final (in 15uL)	Volume
Tris-HCl pH 7.4 (or Tris acetate 7.6)	1000	mM	10	mM	0,10	µl
DMF	100	%	10	%	1,50	µl
Pitstop	1000	uM	70	uM	1,05
MgCl₂ (or Mg acetate)	1000	mM	5	mM	0,05	µl
Nuclease-free Water					4,30	µl
Total Volume					7	µl


Loading buffer w/o SBS	Stock conc		Final conc		V(µl)
Tris-HCl pH 7.4	1000	mM	10	mM	5
DMF	100	%	10	%	50
MgCl2	1000	mM	5	mM	2,5
NaCl	5000	mM	20	mM	2
Glycerol (filtered)	50	%	10	%	100
Nuclease-free Water					340,5
Total Volume					500


Final Loading buffer	Stock conc		Final conc		V(µl)
Loading buff w/o SBS					95
short SBS oligo	100	uM	5	uM	5
Total Volume					100


Name	i7 index primer	Reverse complement index
s3_i7_S701	CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT	TAAGGCGA
s3_i7_S702	CAAGCAGAAGACGGCATACGAGATCTAGTACGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT	CGTACTAG
s3_i7_S703	CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT	AGGCAGAA
s3_i7_S706	CAAGCAGAAGACGGCATACGAGATCATGCCTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT	TAGGCATG
s3_i7_S707	CAAGCAGAAGACGGCATACGAGATGTAGAGAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT	CTCTCTAC
s3_i7_S708	CAAGCAGAAGACGGCATACGAGATCCTCTCTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT	CAGAGAGG
s3_i7_T267	CAAGCAGAAGACGGCATACGAGATTACTCTTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT	GAAGAGTA
s3_i7_T268	CAAGCAGAAGACGGCATACGAGATAACTAACCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT	GGTTAGTT
s3_i7_T269	CAAGCAGAAGACGGCATACGAGATTCCTCAATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT	ATTGAGGA
s3_i7_T271	CAAGCAGAAGACGGCATACGAGATGCTGCTTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT	GAAGCAGC
s3_i7_T272	CAAGCAGAAGACGGCATACGAGATACCGAATCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT	GATTCGGT
s3_i7_T273	CAAGCAGAAGACGGCATACGAGATCTCCAGAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT	CTCTGGAG
s3_i7_T274	CAAGCAGAAGACGGCATACGAGATTGCTAGTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT	GACTAGCA
s3_i7_T275	CAAGCAGAAGACGGCATACGAGATCGACCTGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT	GCAGGTCG
s3_i7_T276	CAAGCAGAAGACGGCATACGAGATAGCCAGCCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT	GGCTGGCT
s3_i7_T278	CAAGCAGAAGACGGCATACGAGATGGTATGGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT	TCCATACC

A	B	C
Number of Nuclei per Well	Required Nuclei Concentration (Nuclei/μl)	Approx. Final Output of Well
20,000	4,000	5,000
25,000	5,000	6,250
30,000	6,000	7,500
35,000	7,000	8,750
40,000	8,000	10,000
45,000	9,000	11,250
50,000	10,000	12,500

Public workspaceScale-scATAC

Scale-scATAC