GSM2412284: DMSO; Homo sapiens; ATAC-seq
Cell pellets were re-suspended in 50μl lysis buffer (10mM Tris-HCL pH7.4, 10mM NaCl, 3mM MgCl2, 0.1% IGEPAL CA-630) and nuclei were pelleted by centrifugation for 10 minutes at 500 x g. Supernatant was discarded and the nuclei were re-suspended in 25μl reaction buffer containing 2μl of Tn5 transposase and 12.5ul TD buffer (Nextera Sample preparation kit from Illumina). The reaction was incubated for 30 minutes at 37ºC and 300rpm, and purified using the Qiagen MinElute Kit. Library fragments were amplified using 1x NEBNext High-Fidelity PCR master mix and 1.25μM of custom PCR primers and conditions (Buenrostro et al., 2013). The PCR reaction was monitored to reduce GC and size bias by amplifying the full libraries for five cycles and taking an aliquot to run for 20 cycles using the same PCR cocktail and 0.6x SYBR Green. The remaining 45ul reaction was amplified for additional cycles as determined by qPCR. Libraries were finally purified using the Qiagen MinElute Kit. Libraries were size selected with AMPure beads (Beckman Coulter) for 200-800 base pair size range and quantified by Q-PCR using Kapa Library Quantification Kit (Kapa Biosystems).