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Sample GSM2992365

Query DataSets for GSM2992365

Status

Public on Feb 09, 2021

Title

WT rep 1

Sample type

SRA

Source name

cerebral cortex

Organism

Mus musculus

Characteristics

background strain: mixed C57BL/6 and Sv129/Sv
tissue: motor plus somatosensory cortex
age: Post natal day 28
genotype: wild type
Sex: mixed

Extracted molecule

polyA RNA

Extraction protocol

Total RNA was purified using spin columns of the RNeasy Mini Kit (Qiagen) according to manufacturer’s protocol.
Library preparation was made with the TruSeq mRNA-seq Stranded v2 Kit sample preparation (Illumina) according to manufacturer’s instructions. One µg total RNA was used for poly(A)-selection and Elution-Fragmentation incubation time was 8 min to obtain 120-210 bp fragments. Each library was barcoded using TruSeq Single Index (Illumina). After library preparation, Agencourt AMPure XP (Beckman Coulter, Inc.) was performed for 200 to 400 bp libraries size-selection (282 nt average final library size). Each library was examined on the Bioanalyzer with High Sensitivity DNA chip (Agilent), quantified on Qubit with Qubit® dsDNA HS Assay Kit (Life Technologies), diluted to 4 nM and then pulled together at equimolar ratio.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

4 mice (3males and 1 female)

Data processing

Base-calling: Base calling was performed on the Illumina analysis platform Basespace Onsite using the Generate FASTQ application with default parameters
Trimming: Raw fastq reads were trimmed using sickle v1.33 (Joshin et al., 2011) with parameters –l 25 –q 20
Alignment: Trimmed fastq reads were aligned to mm10 using STAR v2.5.3a (Dobin et al., 2013) to produce BAM alignment files. Multi-mapped reads and reads with more than 0.08 mismatches per pair relative to read length were discarded
Transcriptome assembly: For each samples transcriptome assembly were performed with Cufflinks v2.2.1 (Trapnell C. et al 2010) using the relative UCSC mm10 GTF file. The output GTF files from each of the Cufflinks analysis and the GTF annotation file were sent to Cuffmerge v2.2.1 (Trapnell C. et al 2010) to amalgamate them into a single unified transcript catalog
Gene counts: Gene counts were calculated with featureCounts v1.4.6-p4 (Liao Y et al., 2014) from a GTF containing known UCSC mm10 genes as well as the novel genes detected by Cufflinks (Cufflinks class code “u”) and alignment files.
Differential gene expression analysis: The R package DESeq2 v1.14.1 (Love MI et al., 2014) was then used to normalize counts and detect the differentially expressed genes (FDR < 0.05). Batch effect between replicates (1,2 and4) was added in the design formula of DESeqDataSetFromMatrix function to model it in the regression step and subtract it in the differential expression test.
Isoform expression analysis: Transcripts expression in fragments per kilobase of exon per million reads mapped (FPKM) were estimated and normalized with Cuffdiff v2.2.1 (Trapnell C. et al 2010) with default parameters from Cuffmerge GTF file result and alignment files
Genome_build: mm10
Supplementary_files_format_and_content: DEseq2 results table for the comparisons WT vs KO in txt format with gene names as rownames and the following columns: DESeq2_log2FoldChange, DESeq2_pvalue, DESeq2_padj (p value adjusted for multiple testing with Benjamini-Hochberg method) , WT_1 WT_3 WT_4 KO_1 KO_3 KO_4 the log2(normalised counts) for each sample; feaureCounts results table with raw counts on known and novel genes per samples

Submission date

Feb 12, 2018

Last update date

May 17, 2021

Contact name

Antoine de Chevigny

E-mail(s)

antoine.de-chevigny@inserm.fr

Phone

+33 631587373

Organization name

INMED INSERM U1249