GSM8262741: D4_DnmtWT_TET1_scrambled_H3K27me3_rep1; Mus musculus; OTHER
RNA extraction from cell pellets was performed using the KingFisher Duo Prime Magnetic Particle Processor and the MagMAX mirVana Total RNA kit, according to the manufacturer's instructions. To measure H3K27me3 enrichment after epigenome editing, we used the original CUT&RUN l protocol with minor modifications. Briefly, 2.5-5 x 105 cells per sample were pelleted and washed twice with wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine containing protease inhibitor). The cells were then incubated for the binding with Concavalin A magnetic beads (Polysciences) by rotating for 10 min at RT. The antibody incubation was carried out overnight on a rotor at 4ºC in 100 µL antibody solution (wash buffer with 0.1% digitonin and 2 mM EDTA) containing 1 µL of αH3K27me3 Ab (Cell Signaling C36B11). Samples were then washed in dig-wash buffer (wash buffer with 0.1% digitonin), and incubated with pAG-Mnase on a rotor for 10 min followed by two more washes. The Mnase reaction was then activated by adding 2 mM CaCl2. After incubating at 0°C for 30 min the reaction was stopped with 1x final concentration of STOP solution (2x: 340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.02% digitonin, 1:200 Rnase A). Chromatin was released by incubating samples at 37°C for 10 min. After centrifuging at full speed for 5 min, the supernatant was collected after incubation on magnet stand. Purified DNA was obtained by phenol/chloroform extraction and precipitated with 100% ethanol by centrifugation. The DNA pellet was washed in 70% ethanol, spun down and air-dried before being resuspended in 35 µl of 5 mM Tris-HCl pH 8.5. The pAG-Mnase plasmid was obtained from Addgene (# 123461), and the protein was purified by the Curiecoretech Recombinant Protein Platform. Total RNA was sent to Novogene Co. for polyA-RNA library constructions and sequencing on an Illumina NovaSeq X (paired-end, 150 nucleotide-long reads). Sequencing libraries were constructed from purified CUT&RUN DNA fragments using the xGen low-input DNA library preparation kit from IDT (Cat. #10009859) following the supplier's instructions. Library quality was assessed using 4200 TapeStation system (Agilent) and sequenced on an Illumina NovaSeq X (paired-end, 150 nucleotide-long reads) by Novogene Co.