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Sample GSM3214305

Query DataSets for GSM3214305

Status

Public on Dec 02, 2019

Title

netrin-1 unc5B MUT rep2 [RNA-seq]

Sample type

SRA

Source name

mouse embryonic stem cells

Organism

Mus musculus

Characteristics

cell line: CGR8
cell type: embryonic stem cells
transfection: ppy-cagg-netrin-ires-puro vector expressing netrin-1 transgene
netrin-1 transgene variation: netrin-1 unc5B MUT

Treatment protocol

mESC are stably transfected with ppy-cagg-ires-puro vector empty or encoding a netrin-1 transgene.

Growth protocol

mESC are grown in serum/LIF conditions.

Extracted molecule

total RNA

Extraction protocol

RNA extracted with Qiagen RNeasy columns.
RNA libraries were prepared for sequencing using standard Illumina protocols.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

CGR8 mESC transfected with an empty ppy-cagg-netrin-ires-puro vector expressing the netrin-1 transgene.

Data processing

Software: All analyses were performed using FastQC version 0.11.5 and fastq_screen version 0.9.5, STAR version 2.5.2b, R version 3.3.2 (2016-10-31), x86_64-apple-darwin13.4.0, Base packages: base, datasets, graphics, grDevices, grid, methods, parallel, stats, stats4, utils,
Read quality: Quality of raw reads was assessed using the FastQC quality control tool (http://www.bioinformatics.babraham.ac.uk/projects/fastqc).
Estimation of Contamination: Sample contamination was assessed using the fastq_screen quality control tool (http://www.bioinformatics.babraham.ac.uk/projects/fastq_screen). Bowtie2 indexes for Mus musculus (mm10) and Homo sapiens (GRCh38) were downloaded from the bowtie website.
Mapping and Summarization: The “primary assembly” Mus musculus genome sequence (release GRCm38.p5) and transcriptome annotations (Ensembl release 87) were downloaded from the GENCODE website (files GRCm38.primary_assembly.genome.fa and gencode.vM12.primary_assembly.annotation.gtf, respectively). Raw read data (fastq files) were mapped to these sequences using STAR (with parameters --outFilterType BySJout, --outSAMtype BAM Unsorted, and --quantMode GeneCounts). This last parameter allows direct conversion of the mappings into gene counts.
Normalization: For the quality control representations, the effective library sizes were first computed using function estimateSizeFactors from package DESeq2. The raw count values were then transformed using a variance stabilizing transformation (function varianceStabilizingTransformation from package DESeq2 with parameter blind=TRUE).
Gene filtering: Genes that did not have more than one count per million counts in at least 2 samples were filtered out. Genes were also filtered if their Ensembl description was either empty, “predicted gene”, “predicted pseudogene” or a “RIKEN”.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: counts_raw.txt: Tab-delimited text file includes raw counts for each sample.
Supplementary_files_format_and_content: counts_normalized.txt : Tab-delimited text file includes normalized counts for the samples.
Supplementary_files_format_and_content: model_fc.txt: Tab-delimited text file includes FC, p-value and adjusted p-value.

Submission date

Jun 25, 2018

Last update date

Dec 02, 2019

Contact name

Fabrice Lavial

Organization name

CRCL

Street address

28 rue Laennec

City

Lyon