title: GSM3593807: mATAC-seq_HCT116_R2; Homo sapiens; OTHER
library_construction_protocol: Omni-ATAC-seq: Cells were trypsinized and subsequently inactivated in cell culture media. Following inactivation, cells were pelleted and resuspended in cold PBS (without Ca++ and Mg++).  Cells were stained with Trypan Blue and counted on a hemocytometer.  Lysis and tagmentation was performed exactly as described in [PMID: 28846090] with modifications to inactivation and size selection.  Briefly, 100,000 cells were lysed on ice for 3 minutes in ice-cold 50uL Lysis Buffer (10mM Tris pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% NP-40, 0.1% Tween20, 0.01% Digitonin in DEPC H2O), resuspended in 1mL ice-cold RBS-Wash (10mM Tris pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% Tween20) and pelleted cold at 500g for 10min.  Tagmentation was performed in 1X Tagmentation Buffer (10mM Tris pH 7.4, 5mM MgCl2, 10% DMF, 33% PBS, 0.1% Tween-20, 0.01% Digitonin) using 100nM Tn5 Transposase for 30 minutes at 37C.  Tagmentation was inactivated with the addition of 5 volumes SDS Lysis Buffer (100mM Tris pH 7.4, 50mM NaCl, 10mM EDTA, 0.5% SDS in DEPC H2O) and 100ug Proteinase K (Invitrogen #25530049) for 30 minutes at 55C followed by Isopropanol Precipitation using GlycoBlue (Invitrogen #AM9516) as a carrier.  DNA was size selected using Ampure XP beads (0.5X – 1.8X). PCR was performed for using Q5 DNA polymerase (NEB #M0491S) with 1X GC buffer (72C 5min, 98C 30sec, 11 cycles of: [98C 10sec, 65C 30sec, 72C 30sec], 72C 5min) followed by a final 1.8X Ampure XP cleanup. methyl-ATAC-seq: Cell lysis was performed identically to Omni-ATAC-seq.  Tagmentation was performed on 250,000 cells using 700nM Tn5 Transposase assembled using pre-annealed methylated-MedsA/B for 30 minutes at 37C.  Inactivation and size-selection was performed identically to our modified Omni-ATAC-seq protocol. Tagmented DNA was End-Repaired for 30min at 37C (5U Klenow Exo- (NEB #M2012S), 1X NEB Buffer 2, and 0.5mM/ea dATP, dGTP, dTTP, and 5mCdCTP (NEB #N0365S)).  End repair was cleaned using a Ampure XP (Beckman Coulter # A63880) 1.8X cleanup.  5% of the product was kept for quality control PCR. Bisulfite conversion was performed using EZ DNA Methylation-Lightning (Zymo #D5030T) following the manufacturer's protocol. PCR was immediately performed using PfuTurbo Cx (Agilent # 600410) (94C 2min, 13 cycles of: [98C 10sec, 65C 30sec, 72C 30sec], 72C 5min) followed by a final 1.8X Ampure XP cleanup.
attributes.tag: GEO Accession
attributes.value: GSM3593807