GSM6755510: PRC2 WT iMEFs H3K27me2 rep2 [D127C74]; Mus musculus; OTHER
Cut&Run-Seq on iMEFs was performed as previously described (Skene et al., 2018). In the case of FL for which a fresh frozen sample was available, ~30 mg of tissue were pulverized in liquid nitrogen, cross-linked with 1% formaldehyde for 8 minutes and treated with 250 mM glycine to stop cross-linking. After 2 washes in PBS, the pellet was incubated successively in two lysis buffers and then suspended in a sonication buffer. Each sample was processed using a Covaris S220 sonicator at peak power 175, duty factor 10 cycle/burst 200, for a duration of 4 minutes. In the case of FL for which only formalin-fixed paraffin-embedded tissue was available, chromatin extraction was performed as described previously (Cejas et al., 2016). 8 sections of 10-µm thickness per sample were deparaffined in xylene 3 times, then progressively rehydrated in ethanol solutions of decreasing concentration. Samples were then incubated at 40°C for 1 hour before treatment for 5 minutes with PK. Reaction was stopped with a Protease inhibitor cocktail. Each sample was then sonicated using Covaris S200 sonicator at peak power 240, duty factor 20, cycle/burst 200, for a duration of 40 minutes. Sonicated samples (~5 µg chromatin/sample) were cleared for 2 h using non-coupled magnetic beads, then further mixed with 5 µL antibody and incubated overnight at 4°C. Complexes were pulled down using Protein A Dynabeads, washed with RIPA buffer and de-cross-linked, and genomic DNA was isolated by standard phenol-chloroform extraction. Cut&Run-Seq libraries were prepared using the Accel-NGS 2S plus DNA library kit (Swift Biosciences) for Illumina barcoded system with 16 cycles for amplification. ChIP-Seq libraries were prepared using the Illumina TruSeq ChIP kit. Both Cut&Run-Seq and ChIP-Seq libraries were sequenced on an Illumina Novaseq 6000 (PE100).