GSM2413402: H3K4me3_F2_Chipseq_6D122 [re-analysis]; Ovis aries; ChIP-Seq
Perirenal adipose tissue was removed from the animals within 10 minutes of euthanasia, flash frozen in liquid nitrogen, stored on dry ice and then stored at -80 o Celcius.  ChIP DNA was isolated briefly as follows:  Frozen tissue was gently pulverised under liquid nitrogen and homogenised in a hand held glass homogeniser in nuclei extraction buffer and nuclei isolated by centrifugation. The isolated chromatin was subsequently treated with micrococcal nuclease and soluble chromatin isolated by mild sonication and centrifugation yielding a relatively homogeneous DNA fragment size of ~ 150bp.  A portion of this isolated soluble chromatin was stored away for each sample and pooled according to its respective group and used as the Input DNA for the INPUT DNA Fetal control libraries.  The remainder of the isolated soluble chromatin  was pre-cleared with Protein A-Sepharose / salmon sperm DNA (Millipore, USA) and then subjected to immunoprecipitation using 10ug of  antibodies specific to, H3K27me3 or H3K27ac or H3K4me3.  Immune complexes were recovered by incubation with Protein A-Sepharose, buffer washes and the DNA- histone protein complexes released  using 1% SDS, 0.1 M NaHCO3, treated with proteinase K, extracted with TE-saturated phenol/chloroform (1:1) and the DNA purified using Minielute DNA columns (Qiagen). The purified DNA (20-100 ng) was quantified using Quant-IT PicoGreen and  validated for purity and size using a High Sensitivity DNA chip run on an Agilent 2100 Bioanalyser (Agilent Technologies). Illumina TruSeq ChIP-Seq Sample Prep Kit  was used according to Illumina recommendations with 10ng  of isolated  immuno-purified nucleosomes to generate single end read libraries for sequencing. ChIP-Seq  50 bp single end read libraries for the samples were run on either an Illumina GA11X or HiSeq20000 sequencing platform.  Input DNA libraries was  run independently on a single flow cell per library on an Illumina GA11X sequencer . H3K27ac and H3K4me3  were run randomly 2 or 4/flowcell on 5 flowcells of an Illumina HiSEQ2000 sequencer.
GEO Accession
GSM2413402