GSM8119387: C16_repB; Homo sapiens; ATAC-seq
hiPSC were collected at Day1 of the DE diff protocol (STEMDiff) and were processed following Salehin et al. 2020. Briefly, 5 x 104 cells were collected at Day 1 of DE differentiation and lysed in cold lysis buffer (10 mM Tris·Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% (v/v) Igepal CA-630). Intact nuclei were separated by centrifugation at 500xg and immediately digested in transposase mix containing 25μl 2x Tagment DNA buffer, 2.5μl Tagment DNA enzyme I (Illumina) and 22.5μl nuclease-free water for 30min at 37°C. Digested chromatin fragments were then purified using the MinElute PCR Purification Kit (Qiagen), according to manufacturer's instructions. The fragments of DNA were then pre-amplified by adding 10μL purified DNA sample, 10μL RNase-free water, 2.5μL of each primer (Supplementary Table 3, where each reaction had non-barcoded primer "Ad1_noMix" and one barcoded primer 'Ad2.1' - 'Ad2.9' added) and 25μL NEBNext High-Fidelity 2x PCR Master Mix (NEB) and was run under the following conditions: 72°C for 5min, 98°C for 30secand then 5 cycles of 98°C for 10sec, 63°C for 30sec and 72°C for 1min. The number of additional cycles to run was calculated by running a RT-qPCR side reaction - a reaction mixture containing 5μL of the pre-amplified PCR product, 3.9μL nuclease-free water, 0.25μL of each primer, 0.6μl 25x SYBR Green and 5μL NEBNext High-Fidelity 2x PCR Master Mix was run under the following conditions: 98°C for 30secand then 20 cycles of 98°C for 10sec, 63°C for 30sec and 72°C for 1min. The linear fluorescence versus cycle number was plotted and the cycle number (N) required to reach one-third of the maximum relative fluorescence was determined. The final amplification reaction (the remaining 45μl pre-amplified PCR product) was run under the following conditions: 98°C for 30sec and then N cycles of 98°C for 10sec, 63°C for 30sec and 72°C for 1min. Amplified samples were then purified using AMPure XP magnetic beads (Beckman Coulter) to remove small fragments and primer-dimers less than 100 bp long (1.3x beads) and large fragments (0.5x beads) using a Dynamag-2 magnet (Thermo Fisher Scientific). To determine the integrity, fragment size and concentration, the DNA library was analyzed using the Agilent HSD5000 ScreenTape System (Agilent). Libraries were then 101 bp paired-end sequenced on an Illumina HiSeq 4000 (Illumina)