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Sample GSM2891766

Query DataSets for GSM2891766

Status

Public on Mar 25, 2018

Title

B216

Sample type

SRA

Source name

Blood sample

Organism

Lynx rufus

Characteristics

ar detection (0=neg;1=pos): 0
tissue: blood

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from 0.5 mL whole blood using the Ambion Mouse RiboPure Blood 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).
Each sample was uniquely tagged with custom index sequences developed at UCLA (Faircloth et al., 2014) comparable to Illumina TruSeq tags. Individual sample libraries were then pooled in equimolar ratios, with 13 or 14 samples per pool and each pool sequenced on two lanes of an Illumina HiSeq 2500 or HiSeq 4000 sequencer

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

regressed_normalized_counts.txt
normalizedcounts.txt
rawcounts_genelength_gccontent.txt

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 > 25 bp).
Alignment : Alignment of reads was performed on TOPHAT2 2.1.0 using the domestic cat (Felis catus) as a reference genome (Ensembl release 85.62; ftp://ftp.ensembl.org/pub/release-82/fasta/felis_catus/dna/Felis_catus.Felis_catus_6.2.dna.toplevel.fa.gz) . To maximize the number of unique reads mapped to the reference genome, we used the following parameters: read mismatches 10, max-insertion-length 12, read-edit-dist 22.
Counts: Aligned reads were converted to raw counts using HTSEQ (Anders et al., 2014) with the “union” mode, resulting in alignment to 21,890 genes.
Normalization: After removal of three globin-related genes (ENSFCAG00000030531, ENSFCAG00000031043,ENSFCAG00000022139) with high expression levels prior to normalization, values for the remaining 21,887 genes were normalized using the trimmed mean of M-values (TMM) method in the edgeR package (Robinson & Oshlack, 2010) in R and adjusted for gene length and GC content using custom Python scripts and the package CQN in R (Hansen et al., 2012).
Differential expression: We performed principal components analysis to identify and remove technical factors from the expression data (Figure S3). Gene by gene linear mixed models were used to identify differentially expressed genes in AR-positive bobcats 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 < 0.05
Genome_build: Felis catus (Ensembl release 85.62)
Supplementary_files_format_and_content: Tab-delimited text file including the regressed normalized counts on 12333 genes.
normalizedcounts.txt
rawcounts_genelength_gccontent.txt

Submission date

Dec 18, 2017

Last update date

Mar 25, 2018

Contact name

Alice Mouton

E-mail(s)

alicemoutonresearch@gmail.com

Organization name

UCLA

Department

Ecology and Evolutionary Biology