ChIP-seq analysis of oligodendrocytes

ChIP-seq assays were performed as described previously (Marie et al., 2018 (link)) using iDeal ChIP-seq kit for Transcription Factors (Diagenode, C01010055). Briefly, O4⁺ MACSorted cells were fixed in 1% formaldehyde (EMS, 15714) for 10 min at RT and the reaction was quenched with 125 mM glycine for 5 min at RT. Lysates were sonicated with a Bioruptor Pico sonicator (Diagenode, total time 8 min) and 4 μg of antibodies were added to sheared chromatin (from 4 million cells for Olig2 and from 1 million cells for histone marks) and incubated at 4°C overnight on 10 rpm rotation. Antibodies used were mouse anti-Olig2 antibody (Millipore, MABN50), rabbit anti-H3K4me3 antibody (Active Motif, 39060), rabbit anti-H3K27Ac antibody (Active Motif, 39034), rabbit anti-H3K4me1 antibody (Ozyme, 5326T), and mouse anti-H3K27me3 antibody (Abcam, ab6002). Mock (rabbit IgG) was used as negative control. Chromatin-protein complexes were immunoprecipitated with protein A/G magnetic beads and washed sequentially according to the manufacturer (Diagenode, C01010055). DNA fragments were then purified using IPure beads v2 (Diagenode, C01010055). Input (non-immunoprecipitated chromatin) was used as control in each individual experiment. The ChIP-seq libraries were prepared using Illumina TruSeq ChIP preparation kit and sequenced with Illumina NextSeq 500 platform.