GSM8214553: H3K27me3_2i; Mus musculus; ChIP-Seq
Native ChIP was performed with 2×10^6 cells. Cells were trypsinized, washed and subjected to hypotonic lysis (50 mM TrisHCl pH 7.4, 1 mM CaCl2, 0.2% Triton X-100, 10 mM NaButyrate, and protease inhibitor cocktail (Roche)) with micrococcal nuclease for 5 min at 37 °C to recover mono- to tri-nucleosomes. Nuclei were lysed by brief sonication and dialyzed into RIPA buffer (10 mM Tris pH 7.6, 1 mM EDTA, 0.1% SDS, 0.1% Na-Deoxycholate, 1% Triton X-100) for 2 hr at 4 °C. Soluble material was incubated with 3 μg of antibody bound to 50 μl protein A Dynabeads (Invitrogen) and incubated overnight at 4 °C, with 5% reserved as input DNA. Magnetic beads were washed as follows: 3x RIPA buffer, 2x RIPA buffer + 300 mM NaCl, 2x LiCl buffer (250 mM LiCl, 0.5% NP-40, 0.5% NaDeoxycholate), 1x TE + 50 mM NaCl. Chromatin was eluted and treated with RNaseA and Proteinase K. ChIP DNA was purified and dissolved in H2O. ChIP-seq libraries were prepared from 5-10 ng ChIP DNA following the Illumina TruSeq protocol. The quality of the libraries was assessed using a D1000 ScreenTape on a 2200 TapeStation (Agilent) and quantified using a Qubit dsDNA HS Assay Kit (Thermo Fisher). Libraries with unique adaptor barcodes were multiplexed and sequenced on an Illumina NextSeq 500 (paired-end, 33 base pair reads). Typical sequencing depth was at least 20 million reads per sample.