title: GSM8756871: MLE12_RNF20_HET_H3K4me3_ChIP_r1; Mus musculus; ChIP-Seq
library_construction_protocol: 107 control, Rnf20+/-, and Rnf20+/-HIF1   KD MLE12 cells were fixed with 1% methanol-free formaldehyde for 10 min at room temperature. 125 mM glycine was added to quench the fixation. After three washes with PBS, cells were suspended in cold PBS at a concentration of 5  106/ml. Equal volume of 2   lysis buffer (100 mM Tris-HCl pH8, 300 mM NaCl, 2% Triton X-100, 10 mM CaCl2) was added for 10 min on ice with gentle shaking. The nuclei were washed with cold PBS and centrifuged at 2500 g, 4   C for 5 min. Chromatin was sheared using a Covaris Ultrasonicator (10% duty factor, 200 cycles, 75 A) for 6 min and the samples were clarified at 16 000g for 15 min. Next, the extracts were pre-cleaned with 75   l protein G beads for 2 h at 4  C, then incubated with 0.5   g Pol II antibody (14958S, Cell Signalling), 1   g Pol II p-Ser5 (ab5131, Abcam), 1   g Pol II p-Ser2 (13499s, Cell Signalling), 1   g H2Bub1 (5546S, Cell Signalling) or 1   g H3K4me3 (ab8580, Abcam) antibodies overnight at 4  C, followed by binding to 75   l BSA-coated protein G beads. Immunoprecipitates were washed twice with low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH7.5, 150 mM NaCl), three times with high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH7.5, 500 mM NaCl), twice with LiCl buffer (10 mM Tris-HCl pH7.5, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 20 mM EDTA) and then eluted with Elution buffer (50 mM Tris-HCl pH8.0, 10 mM EDTA pH8.0, 1% SDS). Samples were treated with RNase A for 1 h at 37   C, followed by Protein K digestion for 2 h at 37   C, and cross-linking was reversed by incubation at 65   C overnight. DNA was purified by using the Qiagen mini elute PCR purification kit (Qiagen, 28004). Libraries were prepared with the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645S/L, E7103S/L) according to the manufacturer's instructions.