GSM8704340: Control4, ChIP; Mus musculus; ChIP-Seq
Differentiated OLs from 4-5 P1 mice were pooled by genotype. At DIV7 cells were washed gently with PBS to remove cellular debris then harvested and pooled using Trypsin A (Sigma-Aldrich, catalog no. T4049). Trypsin activity was neutralized by adding DMEM/F12 supplemented with L-glutamine and 20% FBS. Cells were then centrifuged at 500 rpm for 5 minutes. The pellets were then crosslinked in 1% PFA for 10 minutes at room temperature. To avoid over-fixation, 100 µl of glycine (Cell Signaling, catalog no. 7005S) was added after 10 minutes. The lysates were then centrifuged at 5000 rpm for 5 minutes, and the supernatant was carefully removed. Three washing steps with PBS and centrifugation with the same parameters were then performed. Fresh sonication buffer containing 1% SDS, 50 mM Tris-HCl, pH 8.8, 20 mM EDTA, and a protease inhibitor in DEPC was then added to each sample. DNA shearing was performed by 9 rounds of sonication (Covaris) of 10 pulses each to obtain 200-800 bp DNA fragments. Library preparation and paired-end sequencing using Illumina NovaSeq S1 (100 cycles) was performed at the Crown Genomics institute, Weizmann Institute of Science, Israel.