GSM6964581: ATAC_DNMT_KO_ESC_D0_rep1; Mus musculus; ATAC-seq
ATAC-seq was performed based on published protocols (Buenrostro et al., 2015) using reagents from a Nextera sequencing kit (Illumina, FC-121-1030). Cells were detached using TrypLE Express (Gibco, 12604013), counted, and 10,000 cells aliquoted into DNA LoBind tubes (Eppendorf, 0030108051). Cells were pelleted for 5 minutes at 500g and 4ºC before lysis in 10µL of cold lysis buffer (10mM Tris HCl pH7.4, 10mM NaCl, 3mM MgCl2, 0.1% IgePal CA-630) and immediate repeated centrifugation. Supernatant was removed and the nuclear pellet was suspended in 20µL of transposition mix (1x TD buffer and 0.5µL TDE1 TN5 enzyme) before incubation at 37ºC for 30 minutes with mixing at 1000 RPM using a heated shaker. Samples were purified immediately using a MinElute DNA purification kit (QIAGEN). Transposed DNA was amplified using the adapters described by Buenrostro et al., 2015 at 1.25µM and NEBNext High Fidelity 2x Mastermix (New England Biolabs, M0541) in a 50µL reaction (5 minutes at 72ºC, 30 seconds at 98ºC, followed by 13 cycles of 10 seconds at 98°C, 75 seconds at 65°C and 60 seconds at 72°C). Samples were purified and size selected using Agencourt Ampure XP beads(Fisher Scientific, 10136224) prior to pooling and sequencing.