GSM7697565: ATAC-Sample_3; Homo sapiens; ATAC-seq
ATAC-seq was performed on 1000 CD34+ lineage negative cells. Primary patient cells were sorted directly into 11.25 uL of lysis buffer mix containing 0.25uL 1% digitonin (Promega G9441), 0.25uL 10% Tween-20 (Sigma), 8.25uL of 1X nuclease free PBS (Thermo Fisher AM9625) and 2.5uL nuclease free water (Thermo Fisher 10977049) aliquoted into a well of a 96 well PCR (Thermo Fisher segmented semi-skirted Cat #AB-0900) plate on ice. After sorting, 12.5uL of 2X TD buffer and 1.25uL of Tn5 transposase (Illumina 20034198) were added prior to incubation at 37°C for 30 min. The reaction was stopped using MinElute Reaction Cleanup kit (Qiagen 28204) and the samples eluted in 10uL warmed EB (10mM Tris-HCL, pH8). Samples were then amplified and indexed using NEBNext High-fidelity 2X Mastermix (NEB M0541L) and customised HPLC-purified Nextera indexed primers (IDT) using the following PCR programme: 72°C 5 min, 98°C 30 s, 13 cycles of 98°C 10 s, 63°C 30s, 72°C 1min. Library quality was assessed using High-Sensitivity DNA Kit in a Bioanalyzer instrument (Agilent 5067-4626) and quantitated by qPCR using the NEBNext library quantitation kit (NEB E7630L). Libraries were pooled at 4nM and sequenced on the NextSeq 500 platform (Illumina) using a NextSeq 500/550 High Output v2.5 (75 cycle) sequencing kit (Illumina 20024906), generating 40-bp pair-end reads.