GSM9865740: BJ fibroblast, Global ATAC-see control, added EDTA, bio rep 3; Homo sapiens; ATAC-seq
Following imaging on a Nikon Eclipse Ti2 inverted microscope as described above, HBSS was aspirated and replaced with a reverse-crosslinking solution adapted from (Chen et al., 2016), consisting of 50 mM Tris-HCl, 1 mM EDTA, 1% SDS, 0.2 M NaCl and 250 ug/ml proteinase K. Cells were physically detached using a cell scraper, transferred to microcentrifuge tubes, and incubated overnight at 65°C with agitation at 1,000 rpm in a thermomixer. DNA was subsequently purified using the MinElute Reaction Cleanup Kit (Qiagen, Cat#: 28204) according to the manufacturer's instructions. DNA was amplified using 11, 14, and 17 PCR cycles with NEBNext® HiFi 2× PCR Master Mix (NEB, M0541S) and pre-mixed Illumina DNA/RNA UD Indexes (Set A; cat. no. 20091646), following the PCR protocol described in (Buenrostro et al., 2013). Library quality was assessed by Agilent TapeStation, and products from all three cycle numbers were pooled and purified using 1.8× SPRI beads (Beckman Coulter, cat. no. B23318). Sequencing was performed on an Illumina NextSeq 2000 (P3 flow cell) with paired-end 50 + 50 bp reads and 10 bp dual-indexing (50/10/10/50 cycle configuration).