GSM8972288: wild type, 2HG treated, mBA cells, replicate 1; Mus musculus; ChIP-Seq
Cells per condition were cross linked with 1% formaldehyde for 10 minutes at RT. They were then quenched with 0.125M glycine for 5 minutes and washed with PBS, followed by harvesting in cetrifuge at 4000rpm for 5 minutes. Superntant was discarded and pellets were lysed in lysis buffer (50mM HEPES pH 7.9, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100 supplemnted with protease inhibitors) using Dounce homogenizor, incubation for 20 minutes followed by centrifugation at 4000 rpm for 5 minutes. Nuclei pellets were then washedin wash buffer followed by washing in shearing buffer (0.1% SDS, 1mM EDTA, 10mM Tris pH 8.0). Cells were resuspended in 2ml shearing buffer, split into 6 tubes of 300 ul each and sonicated suing Bioruptor Pico Sonication system (Diagenode) for 70 cycles (30s on, 30 s off) usig the Easy Mode. The library construction started with the end repair step of the protocol. Up to 100ng ChIP DNA was used as starting material. After end repair and A-tailing indexing adapters were ligated. The products were then purified and amplified (12 or 15 PCR cycles) to create the final libraries. After validation (TapeStation; Agilent Technologies) and quantification (Qubit System; Invitrogen) libraries were pooled and the library pool was quantified using the KAPA Library Quantification kit (Peqlab) and the 7900HT Sequence Detection System (Applied Biosystems) The pools were sequenced on an Illumina NovaSeq6000 sequencing instrument using a paired-end 2×100 bp protocol.
GEO
GSM8972288_r1