GSM5061468: H3K4me3_IFNg_Ezh2i_1.5uM_rep1; Mus musculus; OTHER
Cut&Run was performed as previously described (Skene&Henikoff, 2017) with minor adaptations. Primary OPCs were incubated with TrypLE (Gibco #12605010) at 37°C for 5 minutes and collected in cell culture media. Cells were centrifuged for 5 minutes at 300xg and room temperature and then resuspended in wash buffer (20mM HEPES pH 7.5, 150 mM NaCl, 0.5mM Spermidine, 0.01%BSA, 1x Roche Complete Protease Inhibitor tablet). 250 000 cells per condition were centrifuged for 3 minutes at 600xg and room temperature and resuspended in wash buffer. Activated Concanavalin-A beads (Bangs Laboratories BP531) in binding buffer (20mM HEPES pH 7.5, 10mM KCl, 1mM CaCl2, 1mM MnCl2) was added to each condition and incubated for 10 minutes on a rotator at room temperature. Beads with nuclei were now kept on a magnetic stand and washed with Dig-wash buffer (0.05% digitonin in wash buffer). After discarding the liquid, beads were resuspended with primary antibody in antibody buffer (2mM EDTA in Dig-wash buffer) and incubated overnight at 4°C on a nutator. Then beads were washed with freshly prepared Dig-wash buffer and incubated with 2ug/mL Protein A-MNase (Schmid et al., 2004) in Dig-wash buffer for 1 hour at 4°C on a nutator. After two washes with Dig-wash buffer and one wash with low-salt rinse buffer (20mM HEPES pH 7.5, 0.5mM spermidine, 0.05% digitonin) ice-cold incubation buffer (3.5mM HEPES pH 7.5, 10mM CaCl2, 0.05% digitonin) was added to the beads which were subsequently placed in a metal block in an ice-water bath maintained at 0°C for 5 minutes. The beads were then placed on a magnet stand and liquid discarded. Stop buffer (170mM NaCl, 20mM EGTA, 0.05% digitonin, 50ug/mL RNase A, 25ug/mL glycogen, 2pg/mL Yeast spike-in DNA) was added to the beads and incubated for 30 minutes at 37°C. Beads were placed on a magnet stand and supernatant was collected. 2uL of 10% SDS and 2.5uL of 20mg/mL Proteinase K was added to the supernatant and incubated for 1 hour at 50°C. DNA from the samples was purified using the MinElute PCR purification kit (Qiagen) according to manufacturer's instructions. Antibodies were used against H3K27me3 (Cell Signaling 9733S; rabbit; 1ug), H3K4me3 (Diagenode C15410003-50; rabbit; 1ug), H3K27ac (Abcam ab177178; rabbit; 1ug), CTCF (Cell Signaling 3418S; rabbit; 1:100). Sequencing libraries were prepared using KAPA HyperPrep kit (Roche #07962363001) and KAPA Unique Dual-Indexed Adapters (Roche #08861919702) according to manufacturer's instructions, but with the following adjustments. Two post-adapter ligation clean-ups were performed using 0.7x and 1.1x AMPure XP beads respectively. PAGE purification was performed on the post-amplification libraries to remove remaining adapter dimers. Three replicates were performed with primary OPCs obtained from different litters. Libraries were sequenced on the Illumina Novaseq 6000 with a 50-8-8-50 read setup.