title: GSM5182823: WT-4Cell-H2AK119ub1-r2; Mus musculus; ChIP-Seq
library_construction_protocol: The H3K4me3 and H2AK119ub1 CATCH-seq procedure was performed as previously described (Brind'Amour, J. et al., 2015) with some modifications. Counted cells were pelleted and re-suspended in nuclear isolation buffer (10 mM Tris-HCl, pH8.0, 140 mM NaCl, 5 mM MgCl2, 0.6% NP-40, 0.1% Triton X-100, 0.1% deoxycholate, 1 x EDTA-free proteinase inhibitor cocktail and 1 mM PMSF). Chromatin was fragmented for 7.5 or 10 min using 2 or 8 U/μl MNase at 21/25/37 °C (2 U/μl MNase at 21 °C 7.5 min for cultured cells, 2 U/μl MNase at 37 °C 7.5 min for fully grown oocytes and embryos, and 8 U/ul MNase at 25°C 10 min for sperm), and diluted in NChIP immunoprecipitation buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 150 mM NaCl, 0.1% Triton X-100, 1 × EDTA-free protease inhibitor cocktail and 0.1 mM PMSF). In difference to ULI-NChIP, 30 ng modified double-stranded DNAs that can be digested by I-SceI were added into the fragmented chromatin to facilitate the IP process. Chromatin with modified double-stranded DNAs was pre-cleared with 5 of 1:1 protein A:protein G Dynabeads (Life Technologies) and IPed with 700 ng of histone H3K4me3 (15410003, Digagenode) and 650 ng (mESCs, oocytes, and embryos) or 1.95 μg (sperm) H2AK119ub1 (8240, Cell Signaling Technology) antibody–bead complexes overnight at 4 °C. IPed complexes were washed twice with 200 μl of low salt wash buffer (20 mM Tris-HCl, pH 8.0, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 1 x EDTA-free proteinase inhibitor cocktail, 0.1 mM PMSF and 30 ng modified double-stranded DNAs) and twice with 200 μl of high salt wash buffer (20 mM Tris-HCl, pH 8.0, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl, 1 x EDTA-free proteinase inhibitor cocktail, 0.1 mM PMSF and 30 ng modified double-stranded DNAs). Protein–DNA complexes were eluted in 100 μl of ChIP elution buffer (100 mM NaHCO3 and 1% SDS) for 2 h at 65 °C. IPed material was purified by phenol chloroform, ethanol-precipitated and raw ChIP material was re-suspended in 10 mM Tris-HCl pH 8.0. The sequence libraries were generated using the NEBNext Ultra II DNA Library Prep Kit for Illumina (E7645, New England Biolabs) following the manufacturer's instructions, with the exception that we adapted a two-run strategy in the DNA amplification step. The modified double-stranded DNAs were wiped off by I-SceI digestion (5 U/μl I-SceI at 37 °C for 2 h, followed by 20 min heat inactivation at 65 °C) after the first 9 cycles of PCR amplification. Barcoded libraries were pooled and sequenced on the Illumina HiSeq X Ten platform to generate 150 bp paired-end reads.
attributes.tag: GEO Accession
attributes.value: GSM5182823