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Sample GSM2413403 Query DataSets for GSM2413403
Status Public on Nov 09, 2017
Title Fetal_Input_Control [re-analysis]
Sample type SRA
 
Source name Fetal Input Sample
Organism Ovis aries
Characteristics breed: pure bred merino
genotype: wild type
developmental stage: 132 day old fetus
tissue: perirenal adipose tissue
chip antibody: none
molecule subtype: nucleosomal DNA
Treatment protocol No treatment was applied to the animals.
Growth protocol Tissues for this study were provided by Janna L Morrison and Isabella C McMillen from Sansom Institute for Health Research, The University of South Australia, Adelaide, 5001, SA, Australia. All procedures involving animals were carried out with approval from the University of Adelaide Animal Ethics Committee. Animals were maintained on 100% maintenance energy requirement diets and cared for humanely as a research flock according to strict animal ethics guidelines. The ewes for the fetal samples were housed from 110 dpc (days post conception (dpc); term, 150 d) in pens for two weeks before sampling. The pregnant ewes were maintained on a diet that provided 100% of their maintenance energy requirements. Three pregnant ewes were humanely euthanased with an intravenous overdose of sodium pentobarbitone (8.2 g Lethobarb, Virbac Pty ltd, Peakhurst, NSW, Australia) and the uterus removed and fetuses tissue collected at 132±1 dpc.
Extracted molecule genomic DNA
Extraction protocol 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.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina Genome Analyzer IIx
 
Data processing Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then each sample individually mapped to the unmasked OARv3.1.74 sheep genome using the NGS core tool mapping application in CLCBIO (mapping parameters: length fraction =0.7; similarity fraction =0.8; penalties, mismatch =2, insertion =3, deletion =3). H3K27ac samples that had multiple runs on the sequencer were independently merged for each sample and these merged data for each sample were mapped to the genome. Data for each independent sequence run (i.e. not merged) is provided.
Peak calling comparing the H3K4me3 or H3K27ac ChiP-Seq versus the input control was performed using MACS (Zhang et al. 2008). Only peaks found in both replicates per chromatin mark, either H3K4me3 or H3K27ac, were further considered. For H3K4me3 there were 16098 peaks with a FDR 0.1%, whereas for H3K27ac there were 35622 peaks with 1% FDR
Genome_build: OARv3.1
Supplementary_files_format_and_content: 1) xls out of MACS peak calling presenting peak location chr, start, end, length, summit, tags, 10 log(pvalue),fold enrichment, FDR(%) ; 2) bed format showing H3K27ac and H3K4me3 peaks found in both replicates and used in the analysis
 
Submission date Dec 02, 2016
Last update date May 15, 2019
Contact name Tony Vuocolo
E-mail(s) tony.vuocolo@csiro.au
Phone +61732142693
Organization name CSIRO
Street address 306 Carmody Road
City Saint Lucia, Brisbane
State/province Queensland
ZIP/Postal code 4067
Country Australia
 
Platform ID GPL16289
Series (1)
GSE90812 Genome wide chromatin modification associated with brown adipose tissue.
Relations
Reanalysis of GSM1718051
BioSample SAMN06100414
SRA SRX2390402

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data not provided for this record

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