HyDrop-v2-ATAC

Suresh Poovathingal; Florian De Rop; Valerie Christiaens; Koen Theunis; Stein Aerts

Apr 03, 2025

HyDrop-v2-ATAC

DOI

https://dx.doi.org/10.17504/protocols.io.x54v97mmpg3e/v1

Suresh Poovathingal¹,
Florian De Rop^2,3,4,5,
Valerie Christiaens^2,3,4,5,
Koen Theunis^2,3,4,5,
Stein Aerts^2,3,4,5

¹VIB-KU Leuven Center for Brain & Disease Research, CBD Technologies, Single Cell & Microfluidics Expertise Unit, 3000 Leuven, Belgium.;
²Laboratory of Computational Biology, VIB Center for AI & Computational Biology, Leuven, Belgium;
³VIB-KU Leuven Center for Brain & Disease Research, Leuven, Belgium;
⁴Department of Human Genetics, KU Leuven, Leuven, Belgium;
⁵Aligning 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.x54v97mmpg3e/v1

Protocol Citation: Suresh Poovathingal, Florian De Rop, Valerie Christiaens, Koen Theunis, Stein Aerts 2025. HyDrop-v2-ATAC. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v97mmpg3e/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: March 27, 2025

Last Modified: April 03, 2025

Protocol Integer ID: 125622

Keywords: ASAPCRN, scATAC, scATAC-seq, atac-seq, atac, tn5, hydrogel, hydrop, hydrop-atac, droplet, microfluidic, microfluidics, transposase, assay for transposase accessible chromatin, single-cell, atac this protocol, hydrop v2, bulk atac tagmentation, preparation of nuclei, pcr1 cleanup, hydrop run, purification, library purification, nuclei, protocol, preparation

Funders Acknowledgements:

Aligning Science Across Parkinson’s

Grant ID: ASAP-000430

Aligning Science Across Parkinson’s

Grant ID: ASAP-025179

Abstract

This protocol details the preparation of nuclei, bulk ATAC tagmentation, microfluidics setup, HyDrop run, PCR1 cleanup, library purification, and quality control.

Attachments

HyDrop-v2-ATAC.xlsx

29KB

Materials

Preparations:

Experimental parameters:
 
ABCDE
ParameterValue
Occupancy0.6
total nuclei to recover7000nucleidesired recovery
total nuclei to load11666.67correction for occupancy
total nuclei to load12833.34nucleiincludes losses
PCR volume110µLthis includes 25% extra
bead volume45.8µLthis includes 25% extra
Total emulsion vol155.8
nuclei in PCR mix116.67nuc/µL
nuclei in droplets82.36nuc/µLConcentration impacts doublet rate!
cellflow600µL/hrinDrop: 40 nuclei/uL
oilflow700µL/hr10x: 100 nuclei/uL
beadflow250µL/hr
c_ratio0.70588235Ratio impacts PCR concentrations!
pcr1_cycles12cycles
pcr2_cycles12cyclesMay be adjusted
Tagmentation time60min.
Tagmentation temp37C
 
Reagents to be thawed: µ

AB
ThawLocation
2% Digitonin-20
30% BSA-20
PBS4
5X HF Buffer-20
 
Bulk ATAC tagmentation (1:30)

ATAC TD Buffer (ATD) 

ABCDE
4X ATAC TDVol (uL)StockFinal
dH2O135  
DMF100100%40%
Tris-HCl (pH 7.4)10100040mM
MgCl25100020mM
 250  
 
ATAC Reaction Mix (ARM)

ABCDE
ATAC Reaction MixVol (uL)StockFinal
4X ATAC TD3.75400%100%
PBS5.7  
Nuclei in PBS/BSA   
Tn5362.512.5ng/µL
Pitstop1.05100070µM
Tween 200.752.00%0.10%
Digitonin0.750.20%0.01%
 15 
 
Nuclei loading

PCR mix:

ABCDEF
PCR mixVol (uL)StockIn PCR mixIn droplet
Phusion HF Buffer31.17500%142%100%
dH2O27.28   
Optiprep16.5100%15% 
dNTPs6.25251.421mM
Nuclei15   
DTT3.924008560mM
Phusion HF Polymerase3.8520.070.05U/µL
Deep Vent Polymerase3.8520.070.05U/µL
ET SSB2.20.50.010.01U/µL
 110   
 
HyDrop run
 
HyDrop run with flow rates:

ABCD
Flow ratesPriorStable
Cells250600µL/hr
Oil250750µL/hr
Beads250250µL/hr
  
 
PCR program:

ABC
TempTimesample A
72C 20 mins
98C3 mins
98C 10 s|
55C 30 sx 12
72C 1 min|
72C 5min
4Cinf
 
PCR1 cleanup and library purification

GITC Buffer:

ABCDE
GITC BufferVol (uL)StockFinal
GITC833.3360005000mM
dH2O66.67 
EDTA5050025mM
Tris-HCl (pH 7.4)50100050mM
 1000 
 
Clean up mix:
 
ABCDE
Dyna GITC MixVol (uL)StockFinal
GITC buffer180 
Dynabeads10 
DTT102400120mM
 200 
 
EB-DTT-TW Buffer:

ABCDE
EB-DTT-TweenVol (uL)StockFinal
EB490 
DTT5240024mM
Tween 20510%0.10%
 500 
 
Final PCR amplification and library purification 

PCR Mix:

ABCD
PCR 2 mixVol (µL)StockFinal
KAPA50200%100%
Library40 
i75201
Universal P5 partial5201
 100 
 
 PCR amplification:

ABC
TempTime
9503:00
9800:10|
6300:30x 12
7201:00|
7201:00
4Hold
 
Fresh 80% EtOH:
	
ABCD
80% EtOHVol (µL)StockFinal
EtOH8000100%80%
dH2O2000  
 10000 
 

Troubleshooting

Preparations

 Evaluate the experimental parameters below
  
ABCDE
ParameterValue
Occupancy0.6
total nuclei to recover7000nucleidesired recovery
total nuclei to load11666.67correction for occupancy
total nuclei to load12833.34nucleiincludes losses
PCR volume110uLthis includes 25% extra
bead volume45.8uLthis includes 25% extra
Total emulsion vol155.8
nuclei in PCR mix116.67nuc/uL
nuclei in droplets82.36nuc/uLConcentration impacts doublet rate!
cellflow600uL/hrinDrop: 40 nuclei/uL
oilflow700uL/hr10x: 100 nuclei/uL
beadflow250uL/hr
c_ratio0.70588235Ratio impacts PCR concentrations!
pcr1_cycles12cycles
pcr2_cycles12cyclesMay be adjusted
Tagmentation time60min.
Tagmentation temp37C
 

Thaw the following reagents and keep TemperatureOn ice  .
 
AB
ThawLocation
2% Digitonin-20
30% BSA-20
PBS4
5X HF Buffer-20
 

Put centrifuges at Temperature4 °C   and heat block at Temperature65 °C  .
 

Reserve deep well PCR block.
 

Nuclei preparation

Prepare nuclei with your nuclei extraction protocol of choice

Resuspend nuclei in PBS/BSA (0,04% BSA).

Bulk ATAC tagmentation (1:30)

Pre-heat block at Temperature37 °C  .											

Perform nuclei count.						

Meanwhile, prepare ATAC TD Buffer (ATD) and ATAC Reaction Mix (ARM).

ABCD
4X ATAC TDVol (uL)StockFinal
dH2O135  
DMF100100%40%
Tris-HCl (pH 7.4)10100040 mM
MgCl25100020 mM
 250  
 
ABCD
ATAC Reaction MixVol (uL)StockFinal
4X ATAC TD3.75400%100%
PBS5.7  
Nuclei in PBS/BSA   
Tn5362.512.5 ng/uL
Pitstop1.05100070 uM
Tween 200.752.00%0.10%
Digitonin0.750.20%0.01%
 15 
 

Pippette the ARM mix gently to mix all the content.									

Incubate the ATAC Reaction Mix for Duration01:00:00   at Temperature37 °C   without shaking.		

Microfluidics Setup

Turn on pumps and microscope lamp.

Create new directory for pictures of the run and change picture output path.

Take 1x 3 mL syringe and fill with droplet oil. Connect tubing, put in oil pump and prime.

Take 1x 3 mL syringe and fill with mineral oil. Connect tubing and pipet tip and prime.

Take 1x 3 mL syringe and fill with silicone oil. Connect tubing and pipet tip and prime.

Load the beads and put in the bead slot.

Place chip and connect outlet tubing.

Place cold block or ice box and PCR tubes in position.

Prepare program on PCR block.						

Nuclei loading

Prepare the PCR mix as below (add PCR enzymes and nuclei only after other ingredients are mixed).

ABCDE
PCR mixVol (uL)StockIn PCR mixIn droplet
Phusion HF Buffer31.17500%142%100%
dH2O27.28   
Optiprep16.5100%15% 
dNTPs6.25251.421 mM
Nuclei15   
DTT3.924008560 mM
Phusion HF Polymerase3.8520.070.05 U/uL
Deep Vent Polymerase3.8520.070.05 U/uL
ET SSB2.20.50.010.01 U/uL
 110   
 

HyDrop Run

Perform HyDrop run with following flow rates:
 
ABC
Flow ratesPriorStable
Cells250600 uL/hr
Oil250750 uL/hr
Beads250250 uL/hr
 

After the run, perform the linear amplification using the PCR program below:

if emulsion comes above border of PCR, take away oil from below

ABC
TempTimesample A
72C 20 mins
98C3 mins
98C 10 s|
55C 30 sx 12
72C 1 min|
72C 5min
4Cinf
 

PCR1 cleanup and library purification

24m 30s

Thaw GITC Buffer at Temperature65 °C   for Duration00:10:00   and bring to TemperatureRoom temperature  , check if precipitate is gone.				 

If none is available in freezer, prepare GITC Buffer as below.	
 
ABCD
GITC BufferVol (uL)StockFinal
GITC833.3360005000 mM
dH2O66.67 
EDTA5050025 mM
Tris-HCl (pH 7.4)50100050 mM
 1000 
 				

10m

Make clean up mix:
 
ABCD
Dyna GITC MixVol (uL)StockFinal
GITC buffer180 
Dynabeads10 
DTT102400120 mM
 200 
 

Prepare EB-DTT-TW buffer:		
 
ABCD
EB-DTT-TweenVol (uL)StockFinal
EB490 
DTT5240024 mM
Tween 20510%0.10%
 500 
 			

Add Amount125 µL   of recovery agent, gently invert 10 times.

Incubate for Duration00:01:00   and spin down the tubes.					

Discard recovery agent from bottom of tubes.

Add Amount200 µL   of the cleanup mix using P200 pippette.

Incubate the mix for Duration00:10:00   with mixing every 5 minutes.

10m

Pull beads to the sides on magnetic rack and remove supernatant.

Add Amount300 µL   of 80% EtOH for Duration00:00:30   without disturbing pellet.

30s

Remove supernatant and repeat the above 80% EtOH wash one additional time.

Air-dry dry magnetic pellet for no more than Duration00:01:00  .

Spin down pellet and resuspend in Amount50.5 µL   of the EB-DTT-Tween.

Incubate for Duration00:02:00  .

Pull beads to the sides on magnetic rack, transfer Amount50 µL   of the elute to a new PCR tube.

Add 1.2X volume of Ampure beads (Amount60 µL  ).

Perform the purification as recommended by the manufacturer.

Resuspend in Amount40.5 µL   of EB-DTT and take Amount40 µL   eluate to the next step.					

Final PCR amplification and library purification

10m 30s

Prepare the following PCR mix

ABCD
PCR 2 mixVol (uL)StockFinal
KAPA50200%100%
Library40 
i75201
Universal P5 partial5201
 100 
 

Mix the contents well and proceed to amplification:

ABC
TempTime
9503:00
9800:10|
6300:30x 12
7201:00|
7201:00
4Hold
 

After the PCR, add Amount40 µL   of Ampure XP beads (0.4x purification to remove big fragments).

Magnetise and transfer all clear supernatant to a new PCR tube.					 

Add Amount80 µL   of Ampure beads to the clear supernatant and mix well.					 

Incubate the mix for Duration00:05:00  .				 

Prepare fresh 80% EtOH.	
 
ABCD
80% EtOHVol (uL)StockFinal
EtOH8000100%80%
dH2O2000  
 10000 
 				

Pull beads to the sides on magnetic rack.					 
			

Add Amount200 µL   of 80% EtOH for Duration00:00:30   without disturbing pellet.					 
	

30s

Remove supernatant and repeat 80% EtOH wash.					 

Air-dry dry magnetic pellet for no more than 2 minutes.					 

Spin down pellet and resuspend in Amount20.5 µL   of the EB.					 

Incubate for Duration00:05:00  .					 

Pull beads to the sides on magnetic rack and transfer Amount20 µL  to a new PCR tube.	

Quality control

Perform Qbit and Bioanalyzer.					

A	B	C	D	E
Parameter	Value
Occupancy	0.6
total nuclei to recover	7000	nuclei	desired recovery
total nuclei to load	11666.67		correction for occupancy
total nuclei to load	12833.34	nuclei	includes losses
PCR volume	110	µL	this includes 25% extra
bead volume	45.8	µL	this includes 25% extra
Total emulsion vol	155.8
nuclei in PCR mix	116.67	nuc/µL
nuclei in droplets	82.36	nuc/µL	Concentration impacts doublet rate!
cellflow	600	µL/hr		inDrop: 40 nuclei/uL
oilflow	700	µL/hr		10x: 100 nuclei/uL
beadflow	250	µL/hr
c_ratio	0.70588235		Ratio impacts PCR concentrations!
pcr1_cycles	12	cycles
pcr2_cycles	12	cycles	May be adjusted
Tagmentation time	60	min.
Tagmentation temp	37	C

A	B	C	D	E
4X ATAC TD	Vol (uL)	Stock	Final
dH2O	135
DMF	100	100%	40%
Tris-HCl (pH 7.4)	10	1000	40	mM
MgCl2	5	1000	20	mM
	250

A	B	C	D	E
ATAC Reaction Mix	Vol (uL)	Stock	Final
4X ATAC TD	3.75	400%	100%
PBS	5.7
Nuclei in PBS/BSA
Tn5	3	62.5	12.5	ng/µL
Pitstop	1.05	1000	70	µM
Tween 20	0.75	2.00%	0.10%
Digitonin	0.75	0.20%	0.01%
	15

A	B	C	D	E	F
PCR mix	Vol (uL)	Stock	In PCR mix	In droplet
Phusion HF Buffer	31.17	500%	142%	100%
dH2O	27.28
Optiprep	16.5	100%	15%
dNTPs	6.25	25	1.42	1	mM
Nuclei	15
DTT	3.9	2400	85	60	mM
Phusion HF Polymerase	3.85	2	0.07	0.05	U/µL
Deep Vent Polymerase	3.85	2	0.07	0.05	U/µL
ET SSB	2.2	0.5	0.01	0.01	U/µL
	110

A	B	C	D
Flow rates	Prior	Stable
Cells	250	600	µL/hr
Oil	250	750	µL/hr
Beads	250	250	µL/hr

A	B	C
Temp	Time	sample A
72C	20 mins
98C	3 mins
98C	10 s	\|
55C	30 s	x 12
72C	1 min	\|
72C	5min
4C	inf

A	B	C	D	E
GITC Buffer	Vol (uL)	Stock	Final
GITC	833.33	6000	5000	mM
dH2O	66.67
EDTA	50	500	25	mM
Tris-HCl (pH 7.4)	50	1000	50	mM
	1000

A	B	C	D	E
Dyna GITC Mix	Vol (uL)	Stock	Final
GITC buffer	180
Dynabeads	10
DTT	10	2400	120	mM
	200

A	B	C	D	E
EB-DTT-Tween	Vol (uL)	Stock	Final
EB	490
DTT	5	2400	24	mM
Tween 20	5	10%	0.10%
	500

A	B	C	D
PCR 2 mix	Vol (µL)	Stock	Final
KAPA	50	200%	100%
Library	40
i7	5	20	1
Universal P5 partial	5	20	1
	100

	A	B
	Thaw	Location
	2% Digitonin	-20
	30% BSA	-20
	PBS	4
	5X HF Buffer	-20

A	B	C
Temp	Time
95	03:00
98	00:10	\|
63	00:30	x 12
72	01:00	\|
72	01:00
4	Hold

	A	B
	Thaw	Location
	2% Digitonin	-20
	30% BSA	-20
	PBS	4
	5X HF Buffer	-20

A	B	C
Temp	Time
95	03:00
98	00:10	\|
63	00:30	x 12
72	01:00	\|
72	01:00
4	Hold

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HyDrop-v2-ATAC