GSM9025743: PC3 10DPTR, biol rep 3, exp rep 2; Homo sapiens; ATAC-seq
1x105 cells per sample were pelleted and washed twice in cold 1X PBS. Cells were lysed in cold lysis buffer [10mM Tris-HCl, pH 7.5, 10mM NaCl, 3mM MgCl2, 0.1% Tween 20, 0.1% NP40, 0.01% Digitonin, 1% BSA, 100 µM DTT] for 12 minutes on ice. Following lysis, cold wash buffer [10mM Tris-HCl, pH 7.5, 10mM NaCl, 3mM MgCl2, 0.1% Tween 20, 100 µM DTT] was added to each sample and nuclei were pelleted for 10 min at 500 xg at 4 °C. Three biological replicates of each group were prepared, with each containing experimental triplicate samples [Biological replicate sample was split into 3 experimental samples prior to tagmentation/library construction, so each experimental replicate was prepped and sequenced independently]. Transposition/tagmentation was carried out via a modified version of 10X Genomics' ATAC protocol. Briefly, nuclei were resuspended Nuclei Buffer (10X Genomics, #2000207) prior to the addition of ATAC Buffer B (10X Genomics, #2000193) and ATAC Enzyme B (10X Genomics, 2000265). Following gentle mixing, transposition/tagmentation was carried out 37 °C for 1 hr. Tagmented samples were then underwent 14 cycles of PCR amplification with the NEBNext HiFidelity 2X PCR Master Mix (New England Biolabs, #M0541) and Nextera PCR primers. Libraries were purified with 1.2X Ampure XP Beads (Beckman Coulter, #A63881).