Single-nucleus ATAC-seq data generation

Single nucleus ATAC-seq protocol

Nuclei preparation

1. Please select proper nuclei isolation method to generate nuclei suspension, and count with hemocytometer.
2. Adjust samplevolume to 1080 µL--> 9 µL per well (to 96 well plate). Use volume representing 240,000 nuclei(120 wells at 2000 nuclei;please note: lowerlimit for experiment are 100,000 nuclei at this step, 100 wells at 1000 nuclei, in this case reaction volume should be 500 µL --> 5 µL per well (to 96 well plate)).

Tagmentation and sorting

Please note: move plate coolers from -20 to room temperature ~5-10 mins before use

Please note: If libraries for two samples are prepared on the same day in parallel, perform tagmentation in separateplates. Do not mix beforesorting. Sort 20 nuclei from each sampleinto each well of sorting plate.

1. Add 1 µL barcodedTn5 transposomes (0.75 µM, please note: optimal concentration needs to be tested for each new batch of Tn5) per well, e.g. using a BenchSmart™ 96 (Mettler Toledo) and mix 5 times.
2. Incubate at 37°C for 1h with 500 rpm shaking.
3. During tagmentation: Prepare sort plates e.g. using a Biomek i7 Automated Workstation (Beckman Coulter) with 7.5 µL EB (Qiagen), 1 µL barcoded primer i7 (12.5 µM, IDT), 1 µL uni primer i5 (12.5 µM, IDT), 1 µL BSA (200 ng/µL, 1:100 dilution of 2% BSA (A7906, Sigma) in PBS).
4. To inhibit the Tn5 reaction, add 10 µL 40 mM EDTA (final 20 mM; 15575020, Thermo Fisher Scientific) to tagmented samples to quench the reaction and pipette 5x e.g. using a BenchSmart™ 96 (Mettler Toledo).
5. Incubate at 37°C for 15 min with 500 rpm shaking.
6. Add 20 µL 2 x sort buffer (2 % BSA, 2 mM EDTA in PBS), mix 5x and combine in areagent reservoir.
7. Stain nuclei suspension with 3 µM Draq7 (1:100, 7406, Cell Signalling).
8. Sort 20 nuclei per well into 768 wells using a SH800 (Sony).

Fragment release and amplification

1. Shortly spin down sort plates.
2. Add to each well 1 µL 0.2 % SDS (15553035, Thermo Fisher Scientific) and mix e.g. using a Biomek i7 Automated Workstation (Beckman Coulter).
3. Incubate at 55°C for 7 min with 500 rpm shaking.
4. Add 1 µL 12.5% Triton-X and mix e.g. using a Biomek i7 Automated Workstation (Beckman Coulter).
5. Add 12.5 µL NEBNext 2x PCR MasterMix (M0541, NEB) and mix e.g. using a Biomek i7 Automated Workstation (Beckman Coulter).
6. Amplify fragments by PCR. 
    

Temperature profile:

Step 1:                              72°C for 5 min

Step 2:                              98°C for 30 s

[Step 3:                            98°C for 10 s

Step 4:                              63°C for 30s

Step 5:                              72°C for 1 min]

(Repeat steps 3-5 for a total number of 12 cycles) 
Step 6:                             keep at 12°C.

Sample Purification

1. Prepare 3 mL 80% EtOH.
2. Transfer samples to reservoir using 12-channel pipette (mix 5x prior to transfer). Combine wells from all 8 plates -> gives about 16 mL.
3. Transfer ~8mL to one 50 mL Falcon. Add 40 mL PB buffer (19066, Qiagen) including pH- indicator (1:2500) and 2 mL Na-Acetate (3M, pH=5.2) and mix.
4. Transfer on 8 MinElute columns (28004, Qiagen) including 3 mL Extension tube (19587, Qiagen) and apply vacuum.
5. Wash with 700 µL PE (Qiagen) on vacuum station.
6. Spin down 2:30 min with 21,000xg.
7. Transfer column to 1.5 mL LoBind tube (Eppendorf) and apply 20 µL EB (19086, Qiagen) and let stand at room temperature for 2 min.
8. Spin down for 1 min with 15,000xg.
9. Repeat previous two steps with another 20 µL EB (19086, Qiagen) to increase yield.
10. Combine in one 1.5 mL LoBind tube (Eppendorf) -> about 300 µL.
11. Use 160 µL for cleanup with SPRIselect beads (B23318, Beckman Coulter).Add 40 µL EB buffer (Qiagen) to the sample, and add 110 µL SPRIselect beads (B23318, Beckman Coulter) and pipette 10x.
12. Incubate 5 min at room temperature. Separate on magnetic stand.
13. Transfer supernatant to a new tube (around 300 µL).
14. Add 190 ul SPRIselect beads (B23318, Beckman Coulter) and pipette 10x.
15. Incubate 5 min at room temperature. Separate on magnetic stand.
16. Discard supernatant.
17. Wash beads using 500 µL 80% EtOH for 30s (repeat once). Dry beads for 5 min at room temperature.
18. Resuspend beadsin 20 µL EB, pipette10x. Incubate 3 min at room temperature. Separate on magnetic stand.
19. Transfer 19 µL to a 1.5mL LoBind tube (Eppendorf). Add 31 µL EB buffer, 75 µL SPRIselect beads (B23318, Beckman Coulter) and pipette 10x.
20. Incubate 5 min at room temperature. Separate on magnetic stand.
21. Discard supernatant.
22. Wash beads using 500 µL 80% EtOH for 30s (repeat once). Dry beads for 3 min at room temperature.
23. Resuspend beads in 20 µL EB (Qiagen), pipette 10x. Incubate 3 min at room temperature. Separate on magnetic stand.
24. Transfer 19 µL to a LoBind tube (Eppendorf).

Quality control and sequencing

1. Quantify final libraries using Qubit (1 µL/sample, Qubit dsDNA HS Assay Kit (Q32851), Thermo Fisher Scientific) and check library size distribution using 4200 TapeStation (High Sensitivity D1000 ScreenTape (5067-5584) and Reagents (5067-5583), Agilent Technologies).
2. Quantify final library using the PerfeCTa NGS Quantification Kit - Illumina (95154-500, Quantabio).
3. Sequence on a HiSeq4000 or NextSeq500 (Illumina, PE 50 + 10 + 12 + 50 (Read1 + Index1 + Index2 + Read2).

Sequencing primer: