GSM5133618: ATAC H7 rep3; Homo sapiens; ATAC-seq
Library preparation for Circular Chromosome Conformation Capture (4C) was performed as described in (van de Werken et al. 2012) with the exception of initial fixation and lysis procedure, which was adapted from (Miele et al. 2009). 1x107 ARPE or H7 ESCs were dissociated and resuspended in 5ml PBS containing 10% BCS. Cells were crosslinked by adding 5ml of 4% formaldehyde in PBS and incubating at room temperature (RT) for 10 minutes with gentle rocking. Reaction was quenched by adding 2.5ml 2.5M glycine, mixing well and incubating at RT for 5 minutes, then on ice for 15 minutes. Cells were pelleted at 800xg for 10 minutes, resuspended in 1ml of cold Lysis Buffer 1 (10mM Tris pH 8.0, 10mM NaCl, 0.2% IGE-PAL, Protease inhibitor (Roche)) and incubated on ice for 15 minutes. Cells were then dounce-homogenized on ice with a B-type pestle twice for 15 minutes with a minute rest on ice in between. Sample was pelleted at 2500xg for 5 minutes and the pellet was washed with 1x Cutsmart buffer (NEB) and pelleted again. Samples were further processed as described in (van de Werken 2012) using DpnII primary digestion and Csp6I secondary digestion.  ATAC-seq was performed on 5x10^5 ARPE and H7 cells in triplicate as described in (Buenrostro et al. 2015). Sequencing library was prepared as described using Expand Long Template polymerase (Roche) and Illumina adaptor sequence-containing primers unique to the hTERT fragend. ATAC-seq was performed on 5x10^5 ARPE and H7 cells in triplicate as described in (Buenrostro et al. 2015).