GSM5833743: E7_DMSO_d1; Homo sapiens; ATAC-seq
Omni-ATAC was performed as before (Liu et al. 2021) with 70,000 HUDEP-2 cells frozen in 10% DMSO/FBS. On the day of the experiment, cells were thawed on ice and permeabilized with 50 μL RSB buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2) with 0.1% NP40, 0.1% Tween-20 and 0.01% Digitonin for 3 minutes on ice. 1 mL of cold RSB containing 0.1% Tween-20 was added before cells were pelleted again. The pellets were then resuspended with 50 μL transposition mixture (25 μL 2xTD buffer, 2.5 μL transposase from ATAC-seq kit (Illumina), 16.5 μL PBS, 0.5 μL 1% digitonin, and 0.5 μL 10% Tween20), and the reaction was carried out at 37 °C for 45 minutes. Genomic DNA was extracted with a Qiagen MinElute PCR purification kit according to the manufacturer's recommended protocol. Total DNA was used as template for amplification, mixed with 15 μL of 2x Ultra II Q5 mix, 1 μL of 10 μM i7 primer (AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTCAGATGTG), and 1 μL of 10 μM i5 primer (CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTCTCGTGGGCTCGGAGATGT) where the Ns represent barcodes. PCR amplification was performed as follows: 65°C 5 min, 98°C 30 s, 98°C 10 s, 63°C 30 s, repeat step 3-4 for 5-9x, and 63°C 5min. PCR products were purified with AMPure beads and quantified using Qubit and Tapestation before pooling and sequencing. The libraries were sequenced on the NextSeq 500 platform with NextSeq 500/550 High Output Kit v2 (75 cycles). Paired-end sequencing was performed (2x42 bp, 6 bp index).