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Sample GSM4162205

Query DataSets for GSM4162205

Status

Public on Jan 07, 2020

Title

Sample 28: 1417F3L

Sample type

SRA

Source name

Longissimus dorsi muscle

Organism

Sus scrofa

Characteristics

tissue: Longissimus dorsi muscle
age: gestation day 77
Sex: female
genotype: pure Iberian

Treatment protocol

50-100 mg samples of each tissue were ground in liquid nitrogen with mortar and pestle.

Growth protocol

All tissues were collected within 15 min postmortem, snap-frozen in liquid nitrogen, and then stored at -80° C until processing.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from 50-100mg samples of muscle using the RiboPure TM of High Quality total RNA kit (Ambion, Austin, TX, USA) following the manufacturer’s recommendations. RNA was quantified using a NanoDrop-100 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The quality of the RNA was evaluated using the RNA Integrity Number (RIN) value from the Agilent 2100 Bioanalyzer device (Agilent technologies, Santa Clara, CA, USA). The RIN values ranged from 7.8 to 9.8
Sequencing libraries were made using the mRNA-Seq sample preparation kit (Illumina Inc., Cat. # RS-100-0801) according to manufacturer’s protocol. Each library was sequenced using TruSeq SBS Kit v3-HS, in paired end mode with the read length 2x76bp on a on a HiSeq2000 sequence analyzer (Illumina, Inc). Images from the instrument were processed using the manufacturer’s software to generate FASTQ sequence files.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

Sequence reads were analyzed using the Tuxedo protocol (Trapnell et al, 2012).
FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used to assess the quality of raw sequencing data.
TrimGalore (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) was used to quality trim data with default settings and to remove the sequencing adaptors and poly A and T tails (stringency of 6 bp, -s 6) keeping only paired-end reads where both pairs were longer than 40 bp.
Filtered reads were mapped against the pig reference genome (Sscrofa11.1) using TopHat v.2.1.0 (Kim et al., 2013) with Bowtie2 (v.2.2.7.0) applying default settings except that reads were first aligned to the ENSEMBL (11.1.90) transcriptome annotation (-G option), and the distance between both pairs was set to 100 bp (inner-mean distance) and the standard deviation to 150 bp.
Transcripts were assembled using Cufflinks (v2.2.1.), following the protocol proposed by the developer and including the Cufflinks, Cuffmerge and Cuffdiff steps (Trapnell et al., 2012); and transcript abundances were estimated as FPKM (fragments per kilobase of transcript per million mapped reads).
Genome_build: Sscrofa 11.1
Supplementary_files_format_and_content: Processed data file includes normalized expression values (FPKM) for all genes in all 32 samples

Submission date

Nov 15, 2019

Last update date

Jan 07, 2020

Contact name

Cristina Ovilo

E-mail(s)

ovilo@inia.csic.es

Organization name

INIA-CSIC

Street address

Ctra Coruña km 7.5