GSM9225871: unstimulated 3; Homo sapiens; ATAC-seq
For chromatin accessibility profiling of bulk cell populations (Ccd18-Co intestinal fibroblasts), the Omni-ATAC protocol was followed for nuclei isolation and library preparation was followed. Briefly, fibroblasts were collected by trypsin-based detachment and pelleted by centrifugation. After confirming viability of > 95% (Trypan blue hemacytometer), nuclei were isolated by incubating cells for 3 minutes on ice in nuclei isolation buffer containing 0.1% NP-40, 0.1% Tween20 and 0.01% Digitonin. Following washes, nuclei were transposed (Illumina Nextera Tn5 transposase contained in #FC-121-1030) for 30minutes at 37C with 1000rpm mixing. Transposed DNA fragments were isolated using Qiagen MinElute PCR Purification Kit (#28004) and pre-amplified for 5 cycles using NEBNext High Fidelity 2x PCR Master Mix (#M0541) and Illumina Nextera primers (#FC-121-1030). At this stage, the additionally required amplification cycles were determined by SYBR green (Invitrogen #S7563) based quantitative PCR. After amplification for additional cycles (between 3-6), amplified DNA was cleaned up using Qiagen MinElute kit and quantified using KAPA absolute quantification kit (Roche #07960166001). Resulting libraries were quality-controlled on an Agilent TapeStation 4200 using High Sensitivity DNA Screen Tape analysis D1000 (Agilent #5067-5584). Libraries were then pooled and size selected using Ampure XP beads (Beckman # A63880), before re-quantification by TapeStation and adjustment to 2.8 pM and sequencing on an Illumina NextSeq500.