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RNA Sequencing hepatic tissues of Nelore steers

ABSTRACT: Gene expression profiling in hepatic tissues of Nelore steers with divergent profiles for residual feed intake.

PROVIDER: PRJEB7696 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR669163.fastq.gz	Fastqsanger.gz
	ERR669163_1.fastq.gz	Fastqsanger.gz
	ERR669163_2.fastq.gz	Fastqsanger.gz
	ERR669164.fastq.gz	Fastqsanger.gz
	ERR669164_1.fastq.gz	Fastqsanger.gz

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Project description:Growing ruminants maintained under dietary restriction for extended periods will exhibit compensatory growth when reverted to ad libitum feeding. This period of compensatory growth is associated with increased feed efficiency, lower basal energy requirements, and changes in circulating concentrations of metabolic hormones. To identify genetic mechanisms contributing to these physiological changes, 8 month-old steers were fed either ad libitum (control; n = 6) or 60-70% of intake of control animals (feed-restricted; n=6) for a period of 12 weeks. All steers were then fed ad libitum for the remaining 8 weeks of the experiment (realimentation period). Liver was biopsied from each animal at days -14, +1 and +14 relative to realimentation for RNA extraction and gene expression analysis by microarray hybridization. Steers were assigned randomly to one of two treatment groups, control or feed-restricted, and housed indoors in individual pens. Steers were acclimated to their pens for 5 d prior to starting the experimental treatments. Feed was offered once daily between 0630 and 0930 and orts from the previous day's feeding were collected and weighed to estimate actual intake. Control animals were fed ad libitum throughout the 20-wk experimental period. Feed-restricted steers were offered 60-70% of intake of control animals for 12 wks to target a limited rate of gain of approximately 0.5 kg/d. Restricted steers were then fed ad libitum for the remaining 8 wks of the experiment (realimentation period). During the first 3 d of realimentation, feed offered to both treatment groups was divided into two equal rations to gradually adjust restricted animals to full intake. Water was offered ad libitum throughout the experimental period. Approximately 200 mg of liver tissue was collected from each steer by needle biopsy using a Tru-Cut biopsy needle at -14, +1, +14 d relative to realimentation. Liver samples were immediately frozen in liquid nitrogen and stored at -80C until RNA isolation. Total RNA was isolated from 36 liver samples using TRIZOL Reagent (Invitrogen Corp., Carlsbad, CA). Samples were DNase-treated using the TURBO DNA-free kit (Ambion, Inc., Austin, TX) according to manufacturerâ??s instructions, followed by column purification using the RNeasy Mini Kit (Qiagen, Valencia, CA). Quality and concentration of RNA were assessed using a 2100 Bioanlayzer (Agilent Technologies, Palo Alto, CA) and ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Probe labeling, hybridizations of probes to the oligo microarray, and array scanning were performed by the Roche NimbleGen Systems, Inc. Microarray Core Facility in Reykjavik, Iceland according to standard procedures (Madison, WI; http://www.nimblegen.com).

Project description:The objective of this study was to identify differentially expressed genes in the liver of steers with divergent Residual Feed Intake (RFI). Methods:In total 50 purebred Angus, 48 purebred Charolais and 158 Kinsella Composite breed steers were tested for individual feed intake using the GrowSafe system for an average period of 70 to 73 days. During the feedlot test animals were fed at ad libitum with a finishing diet composed of 75% barley grain, 20% barley silage and 5% rumensin pellet. Body weight of each animal was measured at an interval of 28days and ADG for each animal was obtained from a linear regression of serial body weight (BW) measurements (Kgs) on time (days). MWT was calculated as midpoint BW0.75, where midpoint BW was computed as the sum of initial BW of the animal and the product of its ADG multiplied by half the number test days. DMI of each animal was calculated as the average daily feed intake of the animals for the time during the feedlot test (days). The expected DMI for each animal was predicted using the regression intercept and regression coefficients of ADG and MWT on actual DMI, and RFI was computed as the difference between the standardized daily DMI and the expected DMI. At the end of the test, animals were slaughtered and liver tissue was collected immediately after slaughter separately bagged in plastic bags, labeled and flash frozen in liquid nitrogen. The frozen samples were transferred to the laboratory on ice and stored at -80C until RNA extraction. From the frozen samples, 20 samples from each breed were selected for total RNA extraction including six samples with extreme high and six samples extreme low-RFI phenotypes for each breed. Complementary DNA (cDNA) libraries were constructed for each of the 60 animals and consequently single end sequenced using Illumina Hiseq 2500 sequencer. The raw reads were aligned and mapped to the bovine reference genome UMD 3.1 using Tophat2 aligner with default alignment parameters. Thereafter, reads aligned uniquely to each annotated transcript of gene in the bovine genome were counted using HTSeq-count package with default parameters. Read counts from HTSeq-count, sample information (sample id, sire, feed efficiency group and sequencing mode) and gene annotation table from ENSEMBL Biomart were used for differential expression analysis within each breed using the edgeR package in R for the six extreme low-RFI and six extreme high-RFI samples. Results: At a false discovery rate of 0.05 and fold change > 2 , we identified 72, 41 and 175 differentially expressed genes for Angus, Charolais and KC RFI divergent steers respectively.

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RNA Sequencing hepatic tissues of Nelore steers

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