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Sample GSM1198523

Query DataSets for GSM1198523

Status

Public on May 30, 2014

Title

rM5

Sample type

SRA

Source name

muscle

Organism

Macaca mulatta

Characteristics

strain/background: C57BL/6
tissue: muscle
Sex: M
age (years): 10
age (days): 328

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted by a Trizol (Invitrogen) procedure according to the manufacturer's instructions. All RNA quality was assessed using an Agilent 2100 Bioanalyser.
Sequencing libraries were prepared using the TruSeq RNA-Seq Sample Prep Kit (Illumina) according to the manufacturer's instructions.
Polyadenylated RNA was isolated using a poly-dT bead procedure and then chemically fragmented and randomly primed for reverse transcription. After second-strand synthesis, the ends of the double-stranded complementary DNA were repaired. After 3'-end adenylation of these products, Illumina Single-End Sequencing adapters were ligated to the blunt ends of the cDNA fragments. Ligated products were then PCR-amplified (15 cycles). The RNA-seq libraries were sequenced (100 cycles) on the Illumina Hiseq 2000 platform.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

Sample 65
mRNA
Processed data file: 3_species_comparable_gene_RPKM.tsv
Processed data file: 4_species_comparable_gene_RPKM.tsv

Data processing

We built custom exon-junction library for mapping. Human and mouse libraries were based on Ensembl66 annotation, while chimpanzee and rhesus macaque libraries were based on liftovered human annotation.
We used Bowtie2 with a default parameter setting to align the reads to the respective genomes, human version hg19, chimp panTro3, rhesus rheMac2 and mouse version mm9 from the Ensembl66, as well as custom exon-junction libraries.
We kept reads with alignment score > 150, and among those that mapped to two locations, only those with a difference in alignment score >= 5. Among reads that mapped to an exon and exon junction, those with higher scores were kept.
To calculate three species comparable gene expression, only reads in CDS regions which could be recipical liftoverable between human chimpanzee/rhesus macaque were considered. To calculate four species comparable gene expression, which could be recipical liftoverable between human and all other three species were considered.
Genome_build: rheMac2
Supplementary_files_format_and_content: Gene expression was quantified as 1000*P*N/(L-99) where N is the number of reads aligned to liftoverable CDS regions and L is the total length of these regions, P is coefficient used to normalize read number, which equal to mean of read number of all samples divided by read number of current sample.

Submission date

Jul 30, 2013

Last update date

May 15, 2019

Contact name

Jieyi Xiong

Organization name

VIB-KULeuven Center for Cancer Biology

Street address

Herestraat 49, box 912

City

Leuven