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Sample GSM5706049

Query DataSets for GSM5706049

Status

Public on Dec 05, 2021

Title

Hippocampus KO rep4

Sample type

SRA

Source name

Hippocampus

Organism

Mus musculus

Characteristics

tissue: Hippocampus
age (weeks): 18
genotype: PRNP(ZH3/ZH3)

Extracted molecule

total RNA

Extraction protocol

Hippocampi were extracted, flash frozen on dry ice, and RNA was harvested using RNAEasy Mini kit (Qiagen). 500 ng of total RNA was used for poly(A)-mRNA selection using Oligo (dT) magnetic beads and subsequently fragmented to approximately 300bp. cDNA was synthesized using reverse transcriptase (SuperScript II, Invitrogen) and random primers. The second strand of the cDNA incorporated dUTP in place of dTTP.
Libraries were prepared using the TruSeq Stranded mRNA Sample Prep Kit v2 according to the manufacturer's protocol. Briefly, double-stranded DNA was further used for library preparation. dsDNA was subjected to A-tailing and ligation of the barcoded Truseq adapters. All purification steps were performed using AMPure XP Beads. Library amplification was performed with PCR using the primer cocktail supplied in the kit. Final libraries were analyzed using Agilent DNA 1000 chip to estimate the quantity, check the size distribution, and then quantified by qPCR using the KAPA Library Quantification Kit (KapaBiosystems, Merck, Darmstadt, Germany) before amplification Illumina's cBot. Libraries were sequenced 1 x 50bp on Illumina's HiSeq 2500.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Reads were aligned with the STAR mapper to release M14 of the Mus musculus Gencode version of the genome (GRMm38/mm10 assembly) (https://www.gencodegenes.org/mouse/release_M14.html).
A raw count of reads per gene was also obtained with STAR (-quantMode TranscriptomeSAM GeneCounts option). The R/Bioconductor package DESeq2 was used to assess differential expression between experimental groups (Wald statistical test + false discovery rate correction). Prior to processing the differential expression analysis, genes for which the sum of raw counts across all samples was less than two were discarded.
Genome_build: GRMm38/mm10 assembly
Supplementary_files_format_and_content: One report per sample that shows quality of each base, duplication levels, etc
Supplementary_files_format_and_content: Summary per sample (table, the number of reads and the uniquely mapping rates
Supplementary_files_format_and_content: Raw count of reads per gene, as calculated by STAR

Submission date

Nov 28, 2021

Last update date

Dec 05, 2021

Contact name

Arnau Hervera

E-mail(s)

[email protected]

Organization name

IBEC

Street address

Baldiri Reixac 15-21