GSE114655
Global hyperactivation of enhancers stabilizes human and mouse naïve pluripotency [ChIP-seq]
We perform ChIP-seq for RNA Pol II total and phosphorylated forms, in mouse ES cells, and in three culture conditions Overall design: We investigated global regulation of RNA Pol II abundance on chromatin by ChIP-seq in mouse ES cells (E14, BL6/129 mixed background) cultured in three conditions: serum/LIF, 2i, or CDK8/19i.  Similar to published resources of Pol II ChIP-seq in mouse ES cells (ENCODE: https://www.encodeproject.org/; Marks H et al., 2012, Cell; Rahl PB et al., 2010, Cell), we found RNA Pol II, both total and Ser5-phosphorylated, enriched at known transcriptional start sites (TSS) in all three conditions.  ChIP-Seq METHODS: ChIP-seq for RNA Pol II was performed as described by Young and colleagues (Rahl PB et al., 2010, Cell).  Briefly, cells were fixed using 1% formaldehyde, scrape-harvested, resuspended in ChIP lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.1) and sonicated using Covaris water bath sonicator to generate fragments of 150 to 500 bp. Soluble chromatin was diluted 10 fold in ChIP Dilution buffer (1% Triton X-100, 2 mM EDTA pH 8.0, 150 mM NaCl) precleared with Agarose Protein A/G beads (Santa Cruz), and then incubated with antibody specific for total RNA Pol II (N-20, sc-899x, Santa Cruz) or specific for the RNA Pol II Ser5P-phoshorylated form (Abcam #ab5131).  After incubation, immunocomplexes were collected with Agarose Protein A/G beads (Santa Cruz). Next, the immunocomplexes were washed sequentially with Low Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 150mM NaCl), High Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 500mM NaCl), LiCl Wash Buffer (0.25M LiCl, 1% NP40, 1% deoxycholate-Na, 1mM EDTA, 10mM Tris-HCl, pH 8.1) and washed twice with TE (10 mM Tris-HCl pH7.5, 1mM EDTA).  Immunocomplexes were eluted in ChIP elution buffer (1%SDS, 0.1M NaHCO3) and the crosslinking was reverted by incubation at 65 ºC for 8 hrs with 200 mM NaCl. Samples were treated with Proteinase K and RNase A, and DNA was extracted using Phenol-Chloroform.  DNA precipitation was in 100% ethanol with 0.1 M NaAcetate ph5.2 and 2 uLs glycogen (Roche).  The DNA pellet was washed with 70% ethanol, and resuspended in ddH2O. Purified chromatin was used for library construction.  For ChIP-seq the amount of DNA used was ~5 ng from each sample (as quantitated by fluorometry). Samples were processed through subsequent enzymatic treatments of end-repair, dA-tailing, and ligation to adapters as in Illumina's "TruSeq DNA Sample Preparation Guide" (part # 15005180 Rev. C). Adapter-ligated libraries were completed by limited-cycle PCR with Q5 High-Fidelity DNA Polymerase (NEB) and Illumina PE primers (15 cycles), and further purified with a double-sided SPRI size selection to obtain a size distribution in the range of 230-500bp. Libraries were applied to an Illumina flow cell for cluster generation (TruSeq cluster generation kit v5) and sequenced on the Illumina HiSeq4000 with SBS TruSeq v5 reagents by following manufacturer's protocols, to 20-25 million reads per replicate, to a total of >60 million reads per condition.  BIOINFORMATIC METHODS: Sequencing quality for ChIP-seq samples was analyzed with FastQC (Andrews, 2011).  Reads were aligned with Bwa 0.7.5a (Li and Durbin, 2009) to the mouse reference genome (NCBIm37/mm9) using the default seed length (32) and allowing 1 mismatch in the seed.  SAMtools 0.1.16 (Li et al., 2009b) was used to convert the output alignment SAM files to the BAM file format, sort the alignments and eliminate duplicated reads.  BEDTools 2.23.0 (Quinlan, 2014) was used to convert the resulting files to the BED format.  All ChIP and input samples were randomly normalized to the same number of reads.  Peak calling was performed with MACS 2.0.10.20130712 (Feng et al., 2012) using the input sample as control for each one of the ChIP samples.  BigWig files were obtained with bedGraphToBigWig (Kent et al., 2010) from the BedGraph files generated with MACS.  Resulting peaks were annotated with PeakAnalyzer 1.4 (Salmon-Divon et al., 2010) and the distribution of peaks was plotted with SeqMiner 1.3.3e (Ye et al., 2014).