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Sample GSM2086751

Query DataSets for GSM2086751

Status

Public on Mar 31, 2017

Title

F10 RS929

Sample type

SRA

Source name

perirenal adipose tissue

Organism

Ovis aries

Characteristics

breed: pure bred merino
developmental stage: 132 day old fetus
Sex: f
tissue: perirenal adipose
genotype: wild type

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 the Sansom Institute for Health Research, The University of South Australia, Adelaide, 4001, 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 pregnant ewes were housed from 110 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. The pregnant ewes were humanely euthanased with an intravenous overdose of sodium pentobarbitone (8.2 g Lethobarb, Virbac Pty ltd, Peakhurst, NSW, Australia), the uterus removed and fetal tissue collected at 132±1 dpc.

Extracted molecule

total RNA

Extraction protocol

Perirenal adipose tissue was removed from animals within 10 minutes of euthanasia, flash frozen in liquid nitrogen, stored on dry ice and then stored at -80 degrees Celcius. RNA was extracted using Trizol reagent (Life Technologies) and further purified using RNeasy purification (QIAGEN) and treated with DNase1 to remove any contaminating DNA. RNA quantity and integrity were assessed using the Bioanalyzer 2100 (RIN scores >9; Agilent, Santa Clara, USA).
Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 5 ug of total RNA for the construction of paired end read sequencing libraries.
Paired end 100 bp read libraries for the 18 samples were 6-fold multiplexed and run on three flowcells of an Illumina HiSeq2000 sequencing platform. A mean number of reads of ~75.6 (+/-9.8m) million reads was obtained from each library.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

Reads were mapped to the Oarv3.1 reference genome using STAR RNA-Seq aligner (v 2.5.0b) with the following parameters --outFilterMultimapNmax 1 --outFilterMismatchNmax 10 --readMatesLengthsIn Equal --alignIntronMax 100000 --outSAMstrandField intronMotif --outReadsUnmapped Fastx
SNPs were called using the UnifiedGenotyper tool in the Genome Analysis ToolKit (GATK, version 3.4-46-gbc02625), with the following parameters -nt 7 -l ERROR -glm SNP -nda -out_mode EMIT_VARIANTS_ONLY -gt_mode DISCOVERY -stand_call_conf 30 -stand_emit_conf 10 -dt NONE -U ALLOW_N_CIGAR_READS
Filter 1: We removed SNPs that were present in known repeat regions, as described by the UCSC Simple Repeats track (downloaded on 21 February 2014)
Filter 2: SNPs were retained if at least five of the 18 samples had 10 or more reads for both the reference and alternate alleles.
Reads mapping to each SNP were measured and this data used for statistical analysis to identify allele specific expression.
Genome_build: OARv3.1.74
Supplementary_files_format_and_content: tab-delimited text file.
Supplementary_files_format_and_content: Column Descriptor Key:
Supplementary_files_format_and_content: Gene: Gene SNP associated with
Supplementary_files_format_and_content: SNP: SNP coordinate
Supplementary_files_format_and_content: SNPConsequence: DNA consequence of SNP
Supplementary_files_format_and_content: chi_stat: chi test statistic significance of SNP
Supplementary_files_format_and_content: p_val: significance score of SNP
Supplementary_files_format_and_content: HigherAllele: dominantly expressed allele
Supplementary_files_format_and_content: ProportionReadsToRef: proportion of reads mapping to reference genome
Supplementary_files_format_and_content: *_ref : read count for that sample for reference allele
Supplementary_files_format_and_content: *_alt : read count for that sample for alternate allele
Supplementary_files_format_and_content: *_geno : genotype for that sample and SNP as called by GATK

Submission date

Mar 11, 2016

Last update date

May 15, 2019

Contact name

Tony Vuocolo

E-mail(s)

[email protected]

Phone

+61732142693

Organization name

CSIRO