title: GSM5368446: ChIP-seq_YD2_PolIICTD_AB1_CNTRL; Mus musculus; ChIP-Seq
library_construction_protocol: Cerebellar tissues were collected from WT and AtmNes-cre;Aptx-/- mice (10-12 month old) for RNA Pol II  ChIP-seq. Frozen tissues were powdered, suspended in PBS containing 1% PFA to crosslink, pelleted and then washed with ice cold PBS containing protease inhibitor cocktail (Sigma, P8340). Chromatin was extracted in lysis buffer (0.25% Triton X-100, 1mM EDTA pH8.0, 140mM NaCl, 50mM HEPES pH7.9, 10% Glycerol, and 0.5% NP-40) and then washed (in 1mM EDTA pH8.0, 200mM NaCl, 10mM Tris HCl pH8.0, and 0.5mM EGTA pH 8.0). Chromatin was then sheared (in 0.1% SDS, 1mM EDTA pH8.0, and 10mM Tris HCl pH8.0) to generate DNA fragments of 200-500bp using a Covaris M220 sonicator (Covaris). RNA pol II CTD repeat YSPTSPS (phospho S5) antibody (5μg, Abcam, #ab5131) and Magna ChIPTM Protein A+G Magnetic Beads (20μl, Millipore, #16-663) were added to the chromatin and incubated overnight at 4°C. Beads were washed 1x with low salt ChIP wash buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris HCl pH8.0 and 150mM NaCl), 1x with high salt wash buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris HCl pH8.0 and 500mM NaCl) and 1x with LiCl wash buffer (1mM EDTA, 10mM Tris HCl pH8.0, 250mM LiCl, 1% NP-40, and 1% sodium deoxycholate) and finally in TE buffer (1% SDS, 1mM EDTA and 10mM Tris HCl pH8.0). Immunoprecipitated DNA was eluted using (0.1 M NaHCO3/1% SDS) followed by addition of 4μl of 5M NaCl2 per IP to dissociate DNA-protein complexes and incubated at 65°C overnight. For library preparation, immunoprecipitated DNA was quantified using the Quant-iT RiboGreen assay (Life Technologies). Libraries were prepared with HyperPrep  Library Preparation Kits (Roche PN07962363001) and analyzed for insert size distribution on a 2100 BioAnalyzer High Sensitivity kit (Agilent Technologies,) 4200 TapeStation D1000 ScreenTape assay, or Caliper LabChip GX DNA High Sensitivity Reagent Kit (PerkinElmer). Libraries were quantified using the Quant-iT PicoGreen ds DNA assay (Life Technologies) or low pass sequencing with a MiSeq nano kit (Illumina).