title: GSM5260453: RBKO_Input_2; Homo sapiens; ChIP-Seq
library_construction_protocol: Approximately 2 x 106 confluent cells were fixed with 1% formaldehyde for 5 minutes at room temperature under gentle agitation. Formaldehyde was then quenched with the final concentration of 0.125M glycine. Cells were then washed with ice-cold PBS twice, and washed twice in micrococcal nuclease (MNase) buffer (50mM Tris-HCl pH 8.0, 250mM sucrose, 25mM KCI, 1mM MgCl2, 1mM CaCl2, 0.5mM 2-mercaptoethanol, 0.1% NP-40). Nuclei were isolated and DNA was digested with 400 gel units of MNase (NEB cat no. M0247S) for 16 minutes on ice, and digestion was terminated with MNase stop buffer (1% SDS, 50mM Tris-HCl pH 8.0, 20mM EGTA). Nuclei were then sonicated for 2 minutes on low with a bioruptor (30s ON/OFF, Diagenode). Cell debris was removed by centrifugation at 20000g for 15 minutes at 4°C. DNA fragments size was verified via bioanalyzer and most fragments (~90%) were the size of mono or di-nucleosomes (<500 bp). The supernatant was diluted with RIPA buffer without SDS, and 5% of the sample was used as the input for the experiment. Library preparation was performed using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB cat no. E7645S) and NEBNext Multiplex Oligos for Illumina (NEB cat no. 6440S) as instructed by the manufacturer