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Sample GSM2192711

Query DataSets for GSM2192711

Status

Public on Jun 08, 2016

Title

KO rep 2

Sample type

SRA

Source name

cerebellum_KO

Organism

Mus musculus

Characteristics

age: 7-8 weeks
background strain: C57BL/6
genotype: alphaT-catenin KO
tissue: cerebellum

Extracted molecule

total RNA

Extraction protocol

After dissection of the brain, cerebellar tissue (n=3) was used for RNA extraction using TRIzol/chloroform during homogenation of the fatty brain tissue. Subsequently, the Aurum Total RNA mini Kit (Bio-rad) was used according to the manufacturer’s protocol.
Library construction protocol not provided.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

replicate 2
KO_R2

Data processing

The raw data of FASTQ format were processed to remove as much technical artifacts as possible. Low quality ends (phred score <20) were trimmed using the FASTX tool kit (http://hannonlab.cshl.edu/fastx_toolkit). Adapter trimming was performed with cutadapt 1.2.1. Reads shorter than 15 bp after adapter trimming were removed. Quality filtering was done using FastX 0.0.13 and ShortRead 1.16.3. PolyA-reads (more than 90% of the bases equal A), ambiguous reads (containing N), low quality reads (more than 50% of the bases < Q25) and artifact reads (all but 3 bases in the read equal one base type) were removed. The remaining clean reads were aligned against the reference genome of Mus musculus (mus_musculus_GRCm38.73), using the TopHat v2.0.8b software. As parameter options we used: --library-type fr-unstranded –min-intron-length 50 --max-intron-length 500000 –no-coverage-search --no-mixed --read-realign-edit-dist 3. Quality filtering (removal of reads that are non-primary mappings or have a mapping quality < 20), sorting and indexing of the resulting bam-files were done with samtools 0.1.19.
Gene expressions of the transcripts were calculated by counting the number of reads in the alignments that overlap with the gene features, using htseq-count 0.5.4p3. As parameters we took: -m union --stranded=reverse -a 0 -t exon -i gene id. Subsequently, the edgeR 3.12.0 tool was utilized to detect the differentially expressed genes between the alphaT-cat WT and KO samples. The modeling of the variance for each gene was done using both the common dispersion model (the same dispersion value is used for each gene) and the moderated tagwise dispersion model (a distinct, individual dispersion is estimated and used for each gene). EdgeR used with the moderated tagwise dispersion model tends to rank more highly as DE genes that are more consistent in their counts within groups. Finally, genes with a p-value ≤ 0.01, corresponding to a false discovery rate (FDR) value ≤ 0.3, were considered as differentially expressed (DE).
Genome_build: mus_musculus_GRCm38.73
Supplementary_files_format_and_content: tab-delimited text file including raw counts (output HTSeqCount) for each sample. Excel file contains statistical analysis.

Submission date

Jun 07, 2016

Last update date

May 15, 2019

Contact name

Cara J Gottardi

E-mail(s)

c-gottardi@northwestern.edu

Phone

(312) 503-4123

Organization name

Northwestern University