GSM3350483: ATAC_44iN_mD_1; Mus musculus; ATAC-seq
Chromatin: cells were resuspended in 3 ml PBS and crosslinked for 10 min at room temperature with 1% formaldehyde (Sigma F8775). Crosslinking was stopped with 0.125 mM glycine for 5 min at room temperature. 44iN cells (3.107 ) used for TF binding profiling were crosslinked in 3 ml of freshly prepared PBS-DSG 7/17 2 mM at pH 7.0 (Sigma, 80424-5 mg) for 50 min at room temperature with occasional shaking. After pelleting and washing in PBS, cells were incubated for 10 min in 3 ml PBS 1% formaldehyde (Sigma F8775), quenched with 0.125 MM glycine. After fixation, cells were pelleted, washed twice with ice-cold PBS and resuspended in 6 ml of swelling buffer (25 mM Hepes pH 7.95, 10 mM KCl, 10 mM EDTA) freshly supplemented with 1X protease inhibitor cocktail (PIC-Roche, 04 693 116 001) and 0.5% NP-40. After 30 min on ice with occasional shaking, the suspension was centrifuged and resuspended in 450 μl of TSE150 (0.1% SDS, 1% Triton, 2 mM EDTA, 20 mM Tris-HCl pH8, 150 mM NaCl) buffer, freshly supplemented with 1X PIC. Samples were split in 3 (150µL) and sonicated in 1.5 mL tubes (Diagenode) using a Bioruptor Pico (Diagenode) for 7 cycles divided into 30 s ON - 30 s OFF sub-cycles at maximum power, in circulating ice-cold water. After centrifugation (10 min, full speed, 4 °C), the supernatant was stored at −80 °C. Five microlitres was used to quantify the chromatin concentration and check DNA size (typically 200–500 bp). ATAC-seq: 50,000 viable cells were washed with cold 1X PBS, pelleted by centrifugation for 5 min at 500g at 4°C, resuspended in 50 μl of transposition reaction mix (25 μl of Tagmentation DNA buffer, 2.5 μl Tagment DNA enzyme (Illumina) and 22.5 μl nuclease-free H2O) and incubated for 30 min at 37 °C. DNA was purified with a MinElute PCR purification kit (Qiagen). Libraries were prepared as described (Buenrostro et al., 2015) but replacing NEB Next HighFidelity 2X PCR Master Mix, by KAPA HiFi HotStart (KapaBiosystems KM2602) for PCR amplification, after determining the number of cycles needed by qPCR. The concentration and quality of the libraries were assessed as described above. Libraries were sequenced on the HiSeq 2500 sequencer (Illumina) in 65 bases V4 paired-end mode.
GEO Accession
GSM3350483