GSM6281021: ESC H3K9me3 rep2; Mus musculus; OTHER
Total RNA was extracted using an RNeasy micro kit (Qiagen) and treated with DNase (Ambrio, AM1907). cDNA was synthesised from 500ng of RNA with SuperScript II Reverse Transciptase (ThermoFisher Scientific) using random primers. CUT&RUN was carried out according to the EpiCypher® CUTANA CUT&RUN Protocol (v1.6) (https://www.epicypher.com/resources/protocols/cutana-cut-and-run-protocol/). 100,000 ESCs or day 5 NPCs were collected per sample/antibody and washed twice with EpiCypher Wash buffer (20mM HEPES pH 7.5, 150mM NaCl, 0.5mM spermidine) plus protease inhibitors (cOmplete™, Mini, EDTAfree, Roche) before attachment to activated Concanavalin A coated magnetic beads. Beads and cells were resuspended in Antibody Buffer (20mM HEPES pH 7.5, 150mM NaCl, 0.5mM Spermidine, 1x protease inhibitors, 0.01% w/v digitonin, 2mM EDTA) with antibodies (1ug of anti-H3K9me3, anti-H3K27me3 or IgG control; Supplementary Table 1) and incubated overnight at 4°C with gentle rocking.  The next day beads were washed twice in cold Digitonin Buffer (20 mM HEPES pH 7.5, 150mM NaCl, 0.5mM Spermidine, 1x Roche complete protease inhibitors, 0.01% digitonin) and then incubated with Digitonin Buffer plus 2.5μL pAG-MNase (CUTANA™, Epicypher) before addition of 2mM CaCl2 to activate cleavage at 4°C for 2h. The reaction was quenched by addition of 33μl Stop Buffer (340mM NaCl, 20mM EDTA, 4mM EGTA, 50μg/ml glycogen, 50μg/ml RNase A), vortexted and incubated at 37°C for 10minutes to enable the release of DNA fragments. Sample was cleaned using a magnetic rack and the supernatant containing DNA was purified with a MinElute PCR Purification Kit (Qiagen). RNA-seq - 500 ng per sample was processed using KAPA's stranded RNA HyperPrep RiboErase kit and sequenced on a NovoSeq instrument after pooling libraries in equimolar quantities, using a 2x 151 bp paired-end run, resulting in over 15 million reads per sample. Libraries were prepared according to manufacturer's instructions using the following kits: NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® and NEBNext® Multiplex Oligos for Illumina® (New England BioLabs) and pooled in equimolar quantities. Sequencing was carried out on a Novaseq6000 with 150bp PE reads.