GSM5683085: MmES_Cbx2.VD-2x_run1; Mus musculus; ChIP-Seq
ChIP samples were collected from two biological replicates (independent mESC cell lines). For chromatin extraction, 30-50 x 106 cells were trypsinized, washed once in PBS and fixed with 1% formaldehyde for 8 minutes at 25 ºC, followed by reaction quenching with addition of glycine to a final concentration 0.12 M and incubation on ice for 10 minutes. Cells were washed twice in 10 ml ice-cold PBS, followed by centrifugation at 680 × g for 5 min. Cells were resuspended and incubated for 10 min in 10 ml Paro Rinse 1 buffer (10 mM EDTA, 10 mM TRIS, 0.5 mM EGTA) on ice, followed by centrifugation at 680 × g for 5 min. Cells were resuspended in 10 ml Paro Rinse 2 buffer containing 0.25% Triton X-100, 1 mM EDTA, 10 mM TRIS, 0.5 mM EGTA, and 200 mM NaCl, pass three times through a prechilled G26 syringe, and incubated for 10 min on ice followed by centrifugation at 680 × g for 5 min. Final cell lysis was performed with Mnase Digest Buffer (20 mM TRIS, 5 mM MgCl2, 1 mM CaCl2, 10 mM NaCl, 0.25 M Sucrose, 1% Triton X-100, supplemented with protease inhibitor cocktail mix (Roche, 11836170001)) in a concentration-dependent volume (16500 cells per microliter) for 0.5 to 1 hour on ice. Cross-linked chromatin was subjected to MNase digestion (50 Units MNase per 1 ml buffer) in a waterbath at 37°C for 20 minutes with repeated stirring. The digest was stopped by adding 10x Stop Buffer to a final concentration of 150 mM NaCl, 100 mM EDTA, 200 mM EGTA on ice and SDS to 0.1 % final concentration. MNase-digested chromatin was subjected to sonication (four cycles of 32''ON/50''OFF) in a Bioruptor Pico instrument (Diagenode) according to the manufacturer's instructions. Sonicated chromatin was centrifuged at 12,000 × g for 10 min at 4°C and supernatant was used for further steps. For biotin ChIP-seq, 100 µg chromatin, extracted from mESCs expressing biotinylated Cbx.VDs were applied to 30 µl of streptavidin M280 magnetic beads previously blocked for 1 hour at 4ºC with 1% cold fish skin gelatin (Sigma-Aldrich, G7041) and 100 ng yeast tRNA (Thermo Fischer, 15401011) supplemented with protease inhibitor cocktail mix (Roche, 11836170001). Chromatin was incubated with beads at 4ºC, overnight with end-over-end rotation. Following overnight incubations, beads were washed with two rounds of 2% SDS, 1x high salt buffer, 1x LiCl Buffer and two rounds of TE. Beads were treated with RNaseA for 30 min at 37°C, Proteinase K for 3 hours at 50°C then de-crosslinked over night at 55°C. DNA was purified with Phenol-Chloroform extraction and EtOH precipitation. ChIP-seq, libraries were prepared using the NEBNext Ultra II DNA library prep Kit for Illumina (New England Biolabs, E7645L) according to the manufacturer's instructions. Samples tagged with unique index barcodes were combined at equal molar ratios and sequenced as pools on a NovaSeq 6000 sequencing system (Illumina) according to Illumina standards with 100 base-pair single-end sequencing. Library demultiplexing was performed following Illumina standards.
GEO Accession
GSM5683085