title: GSM3080957: Pol II ChIP-seq PTH; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were fixed with 1% formaldehyde for 15 minutes and quenched with 0.125M glycine. Cell pellets were frozen in an ethanol dry ice bath and shipped to Active Motif for FactorPath analysis. The chromatin was isolated from the pellets by adding lysis buffer followed by disruption with a Dounce homogenizer. Lysates were sonicated and the DNA sheared to an average length of 300–500 bp. Genomic DNA (Input) was prepared by treating aliquots of chromatin with ribonuclease, proteinase K and heat for decross-linking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a NanoDrop spectrophotometer. Extrapolation to the original chromatin volume allowed quantitation of the total chromatin yield. An aliquot of chromatin (30 µg) was precleared with protein A agarose beads (Invitrogen, Thermo Fisher Scientific). Genomic DNA regions of interest were isolated using 4-µg antibody against ZNF384 (lot A57874; Sigma HPA004051). Complexes were washed, eluted from the beads with SDS buffer, and subjected to ribonuclease and proteinase K treatment. Cross-links were reversed by incubation overnight at 65°C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. ChIP-seq (Illumina) ChIP and Input DNAs were prepared for amplification by converting overhangs into phosphorylated blunt ends and adding an adenine to the 3'-ends. Illumina genomic adapters were ligated and the sample was size-fractionated (200–300 bp) on an agarose gel. After a final PCR amplification step (18 cycles), the resulting DNA libraries were quantified and sequenced on HiSeq 2000. Sequences (50nt reads, single end) were aligned to the mouse genome (mm10) using the Burrows-Wheeler algorithm. Alignments were extended in silico at their 3'-ends to a length of 150 bp, which is the average genomic fragment length in the size-selected library, and assigned to 32-nt bins along the genome. The resulting histograms (genomic “signal maps”) were stored in BAR and bigWig files. ZFP384 peak locations were determined using the MACS algorithm (v1.4.2) with a cutoff of P = 1e-7.
attributes.tag: GEO Accession
attributes.value: GSM3080957