GSM5825632: scNanoATAC_HFF1; Homo sapiens; ATAC-seq
Cells (100,000-200,000 cells before freezing) were quickly thawed at 37℃ and centrifuged at 500 g for 5 min at 4℃. Then nuclei were isolated with Omni-ATAC lysis buffer (10 mM Tris-HCl (pH 7. 4), 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA630, 0.1% Tween-20, 0.01% digitonin) and incubated on ice for 3-5 min. Nuclei were tagmentated with customized Tn5 transposome which was embed with one adaptor sequence. The reaction was incubated on a thermomixer at 800 rpm, 37℃,30 min and stopped by tagmentation stop buffer. Single nucleus stained with DAPI was flow sorted into each plate well containing lysis buffer and then incubated at 65 °C for 30 min. SDS was quenched with 4μL 10% Tween-20, and PCR was performed using PrimeSTAR GXL DNA Polymerase. Then thermal cycled as follows: 68°C for 10 min, 95°C for 1 min, 16 cycles of 94°C for 15 s, 63°C for 30 s, 68°C for 8 min and finally 68 °C for 5 min. 48 wells with different inner barcodes were pooled and purified with 1X AMPure XP beads. Another four cycles of amplification was carried out in a 50 µL GXL DNA Polymerase system with 1 μM Outer Primer. The amplified products were purified with 1X AMPure XP beads twice. 20 products with different outer barcodes (960 cells in total) were pooled together and sequenced on one MinION flow cell (Oxford Nanopore Technologies; R9.4.1).