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Sample GSM2563475

Query DataSets for GSM2563475

Status

Public on Aug 30, 2017

Title

B4: High Fat-Late Exercise (HF-LEX)

Sample type

SRA

Source name

Femoral mid-diaphysis marrow

Organism

Rattus norvegicus

Characteristics

strain: Sprague-Dawley
age: 120-123 days
Sex: male
tissue: Femoral bone marrow
group: High Fat-Late Exercise (HF-LEX)

Treatment protocol

Eighty male SD rats were randomised at weaning into chow-fed froup (C-SED; n=20) which obtained 18% of their energy from fat (Diet 2018, Harlan Teklad, USA) or a high-fat fed group (n=60) which obtained 45% of their total energy intake from fat (D12451, Research Diets, USA). The high-fat fed group was further divided into three sub-groups. The high-fat sedentary (HF-SED) group which had only spontaneous movement within the cage, the high-fat late-exercise (HF-LEX) group, which was allowed spontaneous activity upto day 60 after which it had access to a running wheel from day 67 to day 120 and the high-fat early exercise (HF-EEX) group which had access to a running wheel from day 22 to day 60 followed by spontaneous movement for the remainder of the experiment.

Growth protocol

Eighty 21-23 days old male SD rats were kept kept in pairs in cages and had ad libitum access to food and water. They were housed in a temperature controlled room (25oC) with a 12 hour light/dark cycle.

Extracted molecule

total RNA

Extraction protocol

The left femur was partially sawn at the junction of (1) the distal and the middle thirds and (2) the middle and proximal thirds.The femur was snapped and the resulting mid-diaphysis was centrifuged (10,000 rpm, 4oC, 30s) to obtain the bone marrow. Total RNA was extracted using TRIzol® and cleaned up using RNeasy minikit. A minimum of 1.5 ug of total RNA was sent to Novogene for RNA-seq
After QC, mRNA is enriched from total RNA by Oligo (dT) beads. The mRNA is fragmented randomly by adding fragmentation buffer, then the cDNA is synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina) , dNTPs, RNase H and DNA polymerase I are added to initiate the second-strand synthesis. Second, after a series of terminal repair, A ligation and sequencing adaptor ligation, the double-stranded cDNA library is completed through size selection and PCR

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

Read quality was determined using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/)
Reads were trimmed (phred score<30 trimmed) using PRINSEQ (http://prinseq.sourceforge.net/) and processed reads that were <50 bases in length were discarded
Reads were aligned to the rat transcriptome (reference sequence NCBI version Rnor_5.0) using TopHat (version 2.1.0). Differential gene expression was determined using Cuffdiff (Cufflinks version 2.2.1). Cuffdiff command was used with parameters -u (multi read correction algorithm) and -b (bias detection and correction algorithm).
Genome_build: Rattus norvegicus NCBI version Rnor_5.0
Supplementary_files_format_and_content: diff file format with following grouping definitions:
Supplementary_files_format_and_content: #G1 - HF-EEX (High-fat early-exercise)
Supplementary_files_format_and_content: #G2 - HF-LEX (High-fat, late exercise)
Supplementary_files_format_and_content: #G3 - HF-SED (High-fat, sedentary)
Supplementary_files_format_and_content: #G4 - Chow-SED (Chow-sedentary)

Submission date

Apr 04, 2017

Last update date

May 15, 2019

Contact name

Justin M. O'Sullivan

E-mail(s)

justin.osullivan@auckland.ac.nz

Organization name

University of Auckland

Department

Liggins Institute