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Sample GSM3014727

Query DataSets for GSM3014727

Status

Public on Mar 02, 2018

Title

Pdgfb_Trp53_ko_1

Sample type

SRA

Source name

Trp53 gRNA PDGFB tumorspheres

Organism

Mus musculus

Characteristics

strain: Ntv-a; Nes-Cre; LSL-Cas9
genotype/variation: Ntv-a T/T; Nes-Cre +/T; LSL-Cas9 +/T
cell type: Trp53 gRNA PDGFB tumorspheres

Treatment protocol

Growth protocol

NeuroCult proliferation kit (Stem Cell Technologies, Cat. 05702), supplemented with 10 ng/ml recombinant human EGF (Gibco, Cat. PHG0313), 20 ng/ml basic-FGF (Sigma-Aldrich, Cat. F0291-25UG), and 1 mg/ml Heparin (Stem Cell Technologies, Cat. 07980).

Extracted molecule

total RNA

Extraction protocol

TRIzol reagent (Invitrogen, Cat. 15596-026).
800 ng of total RNA from the samples was used. PolyA+ fraction was purified and randomly fragmented, converted to double stranded cDNA and processed through subsequent enzymatic treatments of end-repair, dA-tailing, and ligation to adapters as in Illumina's "TruSeq Stranded mRNA LT Sample Prep Kit" (this kit incorporates dUTP during 2nd strand cDNA synthesis, which implies that only the cDNA strand generated during 1st strand synthesis is eventually sequenced). Adapter-ligated library was completed by PCR with Illumina PE primers. The resulting purified cDNA library was applied to an Illumina flow cell for cluster generation and sequenced in paired-end format (Illumina NovaSeq 6000), leading to 101bp paired-end reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

Isolation of poly A+ RNA from total RNA.
Strand-specific RNA-Seq (dUTP).
processed data file: FPKM_table.txt

Data processing

RNA-seq analysis:
RNA-seq data were analyzed with the nextpresso pipeline (http://bioinfo.cnio.es/nextpresso/), as follows: RNA-seq reads were aligned to the mouse genome (NCBI37/mm9) with TopHat-2.0.10 (using Bowtie 1.0.0 and Samtools 0.1.19), allowing three mismatches and twenty multihits. Transcripts assembly, estimation of their abundances were calculated with Cufflinks 2.2.1, using the mouse NCBI37/mm9 transcript annotations from https://ccb.jhu.edu/software/tophat/igenomes.shtml .
Genome_build: NCBI37/mm9
Supplementary_files_format_and_content: FPKM_table.txt: FPKM values calculated with Cuffnorm.

Submission date

Feb 15, 2018

Last update date

Mar 02, 2018

Contact name

Osvaldo Graña

Organization name

CNIO

Department

Structural Biology

Lab

Bioinformatics Unit