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Sample GSM4299911

Query DataSets for GSM4299911

Status

Public on Feb 06, 2020

Title

Gastroc Post 20456 [US.1577086]

Sample type

SRA

Source name

Gastrocnemius muscle

Organism

Macaca mulatta

Characteristics

tissue: Gastrocnemius muscle
animal: 20456
time_point: Post

Extracted molecule

polyA RNA

Extraction protocol

Tissue punch biopsies were taken from adipose and skeletal muscle tissue, snap frozen in liquid nitrogen, and stored at -80 degrees. Samples were then thawed and homogenized in Trizol using a TissueLyser II (Qiagen). RNA was extracted using chloroform and a Qiagen RNeasy Mini Kit following the manufacturer's instructions.
cDNA libraries were constructed with Illumina TruSeq stranded mRNA kit and sequenced on an Illumina HiSeq2000 using paired-end, 50 nucleotide reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

sample name:
US.1577086

Data processing

Sequenced library and read quality was assessed using fastqc.
Sequenced reads were aligned using STAR to the MacaM reference genome. Macaque genes were matched to the closest human homolog, defined as the gene with a symbol that matched the macaque symbol and had a canonical Uniprot entry.
Library quality: samples that had at least 75% of reads uniquely mapped to the genome and at least 10 million total reads were included in the analysis.
Gene filtering: genes that had log2 counts per million of at least 1 in one quarter of libraries were included in the analysis.
Normalization: gene expression counts were normalized within each tissue type using edgeR calcNormFactors to produce the gene_count_matrix processed data table. Counts were further normalized using limma voomWithQualityWeights during differential gene expression analysis.
Sample identity was confirmed by two methods. First, by comparing the expression levels of genes known to be highly expressed in, or specific to, adipose tissue and skeletal muscle (Human Protein Atlas). Second, by comparing SNP variants between sample libraries that came from the same animal: a list of SNPs was compiled from the indexed BAM files of all samples using samtools mpileup and filtered to high confidence, highly expressed SNPs using bcftools filter. Samples were compared using BAM-matcher with the Varscan option. Samples with a fraction of SNPs in common above 90% were from the same animal, whereas samples from different animals had a fraction in common of below 80%.
Genome_build: MacaM Rhesus macaque genome
Supplementary_files_format_and_content: gene_count_matrix.csv, where rows are genes/features and columns are samples. Values are TMM normalized counts (not log2 or cpm transformed). Four samples that did not pass library QC are not included.

Submission date

Feb 06, 2020

Last update date

Feb 08, 2020

Contact name

Sara A Murray

E-mail(s)

zsmy@novonordisk.com

Organization name

Novo Nordisk Research Center Seattle, Inc.

Department

Obesity Research Group