GSM5061444: H3K27ac_Ctr_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. Two or three replicates per condition 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.