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Sample GSM919486

Query DataSets for GSM919486

Status

Public on Jun 25, 2012

Title

1789_2day

Sample type

SRA

Source name

Leukocytes from calves housed with their dam, day 2 post-weaning

Organism

Bos taurus

Characteristics

breed: Continental
developmental stage: calf
tissue: leukocytes
treatment: housed with their dam
day post-weaning: 2
sample type: control

Extracted molecule

total RNA

Extraction protocol

Single-read, 36 bp libraries were prepared for 6 animals per treatment, selected at random, on day 0, 1, 2 and 7 of the study. All 48 libraries were prepared from a starting material of 10 µg of high-quality total RNA. Briefly, total RNA was heated at 65˚C for 5 min to disrupt secondary structures and the mRNA was isolated using 100 µL of Dyna oligo(dT) beads. This step was repeated in order to ensure the removal of genomic DNA, ribosomal RNA and other contaminating RNA. Fragmentation Reagent (Ambion) was used to fragment mRNA prior to synthesis of double-stranded cDNA. cDNA was synthesised using random hexamer primers and SuperScript II reverse transcriptase. Following second strand cDNA synthesis, each sample was purified using a QIAquick PCR spin column. Fragments were end repaired with T4 DNA polymerase and E. coli DNA Polymerase I Klenow fragment to remove 3'-overhangs and fill in 5'-overhangs. In order to prepare the cDNA fragments for adapter ligation, an additional "A" base was added to the 3' end of the phosphorylated DNA fragments using Klenow Fragment 3'-to-5' exo-. Adapters were ligated to the DNA fragments using Quick T4 DNA Ligase at room temperature for 15 min. Following adapter ligation, 10 µL of sample was loaded onto a 2% agarose gel in 1X TAE buffer. The gel was run at 120 V for 70 min. The gel was then visualised on a Dark Reader Transilluminator and a band of gel corresponding to the 250 bp ladder band was excised using a GeneCatcher Disposable Gel Excision tip. The DNA was then purified from this gel band using the QIAquick Gel Extraction kit and eluted in 30 µL of EB solution. PCR reactions were performed on the gel-purified cDNA in a 50 µL reaction using Illumina supplied PCR primers 1.1 and 2.1 and Phusion High-Fidelity DNA polymerase for 15 cycles. Following the PCR step, the final libraries were purified using a QIAquick PCR spin column, quantified with a Qubit Fluorometer and stored at -80 ˚C until sequencing. Library size was confirmed by running 5 µL of each library on a gel and visually inspecting for the sole presence of a 250 bp band. Each library was sequenced in an individual lane on one of eight flowcells using a 36 bp single-end sequencing kit (version 4) at a concentration of 8.5 pM. Sequencing was carried out using an Illumina Genome Analyzer II at the Conway Institute, University College Dublin (Dublin, Ireland).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina Genome Analyzer II

Description

mRNA

Data processing

CASAVA version 1.6 used for base calling and FastQC version 0.9.1 for QC checks.
Alignments to Btau4.0 using Bowtie (version 0.12.7).
Gene counts calculated with HTseq (version 0.4.6p2) and Ensembl v61 of the bovine genome.
EdgeR (version 1.6.12) used for identification of DEG.

The processed data file 'read_counts.txt' is available as a supplementary file from the Series record.
Genome_build: Btau4.0

Submission date

Apr 19, 2012

Last update date

May 15, 2019

Contact name

David J Lynn

E-mail(s)

david.lynn@teagasc.ie

Organization name

Teagasc

Department

Animal & Bioscience