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Sample GSM3097896 Query DataSets for GSM3097896
Status Public on Jun 07, 2023
Title 871P4M
Sample type SRA
 
Source name muscle
Organism Bos taurus
Characteristics rfi: Hi
diet: H2
breed: CH
Treatment protocol Average daily live weight gain (ADG) during the RFI measurement period for each animal was computed as the coefficient of the linear regression of BW (kg) on time (d) by using the GLM procedure of SAS 9.1 (SAS Inst. INC., Cary, NC). Mid-test metabolic BW (MBW) was represented as BW0.75 35 d before the end of the test which was estimated from the intercept and slope of the regression line. Residual feed intake was calculated for each animal as the difference between actual DMI (Dry matter intake) and expected DMI. Expected DMI was computed for each animal using a multiple regression model, regressing DMI on MBW, ADG and mean lumbar BF change. Animals were ranked within breed and diet and following a power analysis (www.biomath.info/power/ttest.htm) the most extreme animals for high RFI (n=12) and low RFI (n=12) were selected for further analysis.
Growth protocol This experiment was conducted as part of larger study designed to examine the within-animal repeatability of intake, growth and feed efficiency between the growing and finishing stages of the lifespan of Charolais (CH) and Holstein Friesan (HF) beef steers fed the same diet [4]. Briefly, one hundred and sixty seven cattle comprised of 90 CH and 77 HF were used in the study. Following a dietary adaptation period, individual dry matter intake (DMI) and growth were measured over three 70 d feeding phases. Mean BW (SD) and age (SD) at the start of the feeding phase were 395 kg (37.8) and 283 d (18.3), and 294 kg (42.3) and 306 d (7.7), for CH and HF, respectively. Corresponding BW at the start of phase 3 and phase 4 were, 519 (38.3) and 441 (39.2), and 675 (49.8) and 611 (49.3), respectively. During feeding phases 1 and 3 (high concentrate 1 and 2) steers were individually offered the same concentrates (860 g/kg rolled barley, 60 g/kg soya bean meal, 60 g/kg molasses, and 20 g/kg minerals and vitamins) ad libitum plus a restricted allowance of grass silage daily. Throughout the interim period (Zero grazed grass) they were offered zero-grazed grass (DM 196 g/kg) ad libitum. The grass herbage was harvested (without chopping) twice daily from Lolium perenne dominant swards using a ‘zero-grazer’.
Extracted molecule total RNA
Extraction protocol Total RNA was isolated from 50mg of biopsy samples using QIAzol (Qiagen, UK). Tissue samples were homogenised in 1mL of QIAzol reagent using a rotor-strator tissue lyser (Qiagen, UK) and chloroform (Sigma-Aldrich Ireland, Dublin, Ireland). RNA was subsequently precipitated and purified using the RNeasy plus Universal kit (Qiagen, UK) according to the manufacturer’s guidelines, which included a step to remove any contaminating genomic DNA. The quantity of the RNA isolated was determined by measuring the absorbance at 260nm using a Nanordrop spectrophotometer ND-1000 (Nanodrop Technologies, Wilmington, DE, USA). RNA quality was assessed on the Agilent Bioanalyser 2100 using the RNA 6000 Nano Lab Chip kit (Agilent Technologies Ireland Ltd., Dublin, Ireland). RNA quality was also verified by ensuring all RNA samples had an absorbance (A260/280) of between 1.8 to 2.0 and RIN (RNA integrity number) values of between 8 and 10 were deemed high quality. Any samples that had a (A260/280) absorbance of less than 1.8 were cleaned using Zymo Research RNA clean & concentrator kit (Cambridge Biosciences, UK). High quality RNA samples were selected for cDNA synthesis.
cDNA libraries were prepared from high quality RNA following the manufacturer’s instructions using the Illumina TruSeq stranded mRNA sample prep kit (Illumina, San Diego, CA, USA). For each sample, 1 μg of RNA was used for cDNA library preparation. Briefly, mRNA was purified from total RNA and subsequently fragmented. First strand cDNA was synthesised using Superscript II Reverse Transcriptase (Applied Biosystems Ltd., Life Technologies) followed by second strand synthesis using components of the Illumina TruSeq RNA sample prep kit. Adaptors were ligated to the cDNA which was subsequently enriched by 15 cycles of PCR. Libraries were validated on the Agilent Bioanalyser 2100 using the DNA 1000 Nano Lab Chip kit. cDNA concentration was assessed using Nanordrop spectrophotometer ND-1000 (Nanodrop Technologies, Wilmington, DE, USA) and samples with >25ng/μl were deemed suitable for further analysis. Following quality control procedures, individual RNAseq libraries were pooled based on their respective sample-specific 6bp adaptors and sequenced at 50bp/sequence single-end read using and Illumina HiSeq 2500 sequencer.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
 
Data processing FASTQC software (v0.11.5) was used to check the quality of the raw sequencing reads.
Input reads were then aligned to the bovine reference genome (UMD3.1) using STAR (v2.5.1).
STAR software was used to calculate the number of sequenced fragments overlapping all protein-coding genes from the ENSEMBLv88 annotation of the bovine genome. The number of counts of reads mapping to each annotated gene from HTSeq was than collated into a single file and used for subsequent differential gene expression.
DESeq2 was applied to identify statistically significant differentially expressed genes.
A false discovery rate (FDR) of <0.1 was applied as a threshold to call genes with differential expression levels.
Genome_build: UMD 3.1
Supplementary_files_format_and_content: zipped fastq files
 
Submission date Apr 13, 2018
Last update date Jun 07, 2023
Contact name Clare Mckenna
E-mail(s) mckenncl@tcd.ie
Organization name Teagasc
Department Animal Bioscience
Street address Dunsaney
City Meath
ZIP/Postal code Co Meath
Country Ireland
 
Platform ID GPL19172
Series (1)
GSE113135 The effect of diet and breed on global gene expression profiles of animals divergent for RFI
Relations
BioSample SAMN08930404
SRA SRX3934593

Supplementary file Size Download File type/resource
GSM3097896_871P4M.star.ReadsPerGene.out.tab.gz 100.0 Kb (ftp)(http) TAB
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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