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Sample GSM3118718

Query DataSets for GSM3118718

Status

Public on Nov 16, 2018

Title

7326_6571

Sample type

SRA

Source name

Blood sample

Organism

Marmota flaviventris

Characteristics

tissue: Blood
dispersal (0=resident;1=disperser): 0

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from 1.0 mL whole blood preserved in 2.5 mL PAXGene blood RNA solution using the PAXGene Blood RNA extraction kit, followed by globin removal using the Ambion GLOBINClear Mouse kit (Life Technologies, Inc). RNA was quantified on the Agilent Bioanalyzer (Agilent Technologies, USA).
cDNA libraries were created using a TruSeq Library Prep Kit v2 (Illumina, Madison, WI) and quantified with the KAPA SYBR® Fast qPCR library quantification kit (Kapa Biosystems Inc., Wilmington, MA). Individual sample libraries were pooled in equimolar ratios with 8 to 10 samples per pool. Each pool was sequenced on a single lane of an Illumina HiSeq 2000 or HiSeq 4000 sequencing platform.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Filtering and quality: Raw sequences were processed using Trim Galore! 0.3.1 to remove Illumina adapters and filter out sequences that did not meet the quality thresholds (q > 20, length > 20 bp).
Alignment : Alignment of reads was performed on TOPHAT2 2.1.0 using the thirteen-lined ground squirrel (Ictidomys tridecemlineatus) as a reference genome (Ensembl release 84; ftp://ftp.ensembl.org/pub/release-84/fasta/ictidomys_tridecemlineatus/dna/Ictidomys_tridecemlineatus.spetri2.dna.toplevel.fa.gz) . To maximize the number of unique reads mapped to the reference genome, we used the following parameters: read mismatches 8, bp gap length 10, read-edit-dist 20.
Counts: Aligned reads were converted to raw counts using HTSEQ (Anders et al., 2014) with the “union” mode, resulting in alignment to 22,389 genes.
Normalization: After removal of nine globin-related genes (ENSSTOG00000006499, ENSSTOG00000020876, ENSSTOG00000010650, ENSSTOG00000024317, ENSSTOG00000025073, ENSSTOG00000020904, ENSSTOG00000022777, ENSSTOG00000010645) with high expression levels prior to normalization, values for the remaining 22,389 genes were normalized using the trimmed mean of M-values (TMM) method and adjusted for gene length and GC content using the package VOOM in R (Law et al. 2014).
Differential expression: We performed principal components analysis to identify and remove technical factors from the expression data (sequencing platform, sequencing lane, extraction date, number of uniquely mapped reads, RIN score, neutrophil count). Linear mixed models were used to identify differentially expressed genes in dispersing marmots using the limma package in R (Ritchie et al., 2015). We adjusted our significance values to account for multiple hypothesis testing using the false discovery rate (FDR) method as implemented in the qvalue package in R (Storey et al., 2015) and selected genes falling below q < 0.1
Genome_build: Ictidomys tridecemlineatus (Ensembl release 84), GenBank Assembly ID GCA_000236235.1
Supplementary_files_format_and_content: Comma separated file (csv) including the regressed normalized counts on 11,381 genes
Supplementary_files_format_and_content: regressed_normalized_counts.txt: VOOM count output after technical variation was regressed out
Supplementary_files_format_and_content: normalized_counts.txt: HTSEQ count output normalized using VOOM
Supplementary_files_format_and_content: raw_counts_genelength_gccontent.txt: raw expression counts quantified using HTSEQ

Submission date

Apr 27, 2018

Last update date

Nov 16, 2018

Contact name

Tiffany Armenta

E-mail(s)

tiffany.armenta@gmail.com

Organization name

UCLA

Department

Ecology & Evolutionary Biology