GSM5025144: HAP1_1_70; Homo sapiens; ATAC-seq
Plate-based snATAC-seq 1)One day before the experiment, prepare the 96-well plates by adding 1µL 2X Lysis Buffer (100 mM NaCl pH 7.4, 100 mM Tris-HCl pH 8.0, 40 µg/mL proteinase K, 0.4 % SDS) to each well. Then add 1µL of 10 µM S5xx/N7xx Nextera Index Primer Mix (5 µM each) (TD202, Vazyme) to each well. Seal the plate and store in -80 ºC. Thaw plates at room-temperature before use. 2)Pre-coat 1.5 mL Eppendorf tubes with 500 µL 0.5% BSA/DPBS. Collect 5×105 cells in DPBS into 1.5 mL Eppendorf tube. Centrifuge at 300 g for 5 min at room-temperature. Wash the cells once with 200µL DPBS, then with 200 µL ATAC resuspension buffer (10 mM NaCl, 10 mM Tris-HCl pH 7.4, 3 mM MgCl2). Centrifuge condition is 600 g, 5 min at 4 ˚C. 3)After two steps wash, resuspend cells in 100 µL ATAC cell lysis buffer (10 mM NaCl, 10 mM Tris-HCl pH 7.4, 3 mM MgCl2, 0.1% Tween-20, 0.1% NP40, 0.01% Digitonin), then add 2.5 µL assembled CASB complex (control group was only resuspended in ATAC cell lysis buffer). Mix thorough by pipette. Incubate on ice for 5 min, then add 950 µL ATAC wash buffer (10 mM NaCl, 10 mM Tris-HCl pH 7.4, 3 mM MgCl2, 0.1% Tween-20) to quench reaction. 4)Centrifuge nuclei at 1000 g for 5 min at 4 ˚C, discard the supernatant by pipette carefully. Resuspend nuclei in 1 mL ATAC wash buffer. Centrifuge again. 5)Resuspend nuclei in 50 µL tagmentation reaction (TD501, Vazyme). Incubate at 37 ˚C on a thermomixer (600 rpm) for 30 min. Then, stop the reaction by adding 50 µL tagmentation stop buffer (20 mM EDTA, 10 mM Tris-HCl pH 8.0). Incubate on ice for 10 min. 6)Add 300 µL 0.5% BSA/DPBS into nuclei. Then add DAPI according to manufacturer's instruction. Use FACS (MA900, Sony) to sort single nucleus into each well of prepared 96-well plates. 7)Set lip temperature of PCR machine to 100 ˚C, then incubate plates at 65 ºC for 15 min. 8)Add 2 µL 10% Tween-20 into each well to quench SDS. Then add 6 µL PCR mix (from TD501, Vazyme). PCR reaction as follow: 72 ˚C 10 min, 98 ˚C 1 min, (98 ˚C 30 s, 63 ˚C 30 s, 72 ˚C 30 s) ×25, 72 ˚C 5 min, 10 ˚C hold. 9)Collect all products into a 15 mL conical centrifuge tube. Purify the product using ZYMO DNA clean & concentrator kit. 10)Perform size