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Sample GSM6738877

Query DataSets for GSM6738877

Status

Public on Dec 01, 2022

Title

Hippocampus WT 10wk replicate 4

Sample type

SRA

Source name

hippocampus

Organism

Mus musculus

Characteristics

tissue: hippocampus
gender: male
age: post natal week 10
genotype: WT
strain: C57BL/6J

Extracted molecule

total RNA

Extraction protocol

Collected tissue was stored in RNAlater solution (Thermo Fisher Scientific). Total RNA was isolated using TRIzol reagent (Thermo Fisher Scientific) following the manufacturer’s protocol. Quality control involved quantification using fluorimetry via RiboGreen assay kit (Thermo Fisher Scientific) and RNA integrity was assessed via capillary electrophoresis using an Agilent BioAnalyzer 2100 to generate an RNA integrity number (RIN).
Library creation was completed using oligo-dT purification of polyadenylated RNA, which was reverse transcribed to create cDNA. cDNA was fragmented, blunt ended, and ligated to barcode adaptors. Libraries were size selected to 320 bp ± 5% to produce average inserts of approximately 200 bp, and size distribution was validated using capillary electrophoresis and quantified using fluorimetry (PicoGreen, Thermo Fisher Scientific) and qPCR. Libraries were then normalized, pooled, and sequenced on an S4 flow cell by an Illumina NovaSeq 6000 using a 150-nucleotide, paired-end read strategy. Libraries were prepared and sequenced in two batches based on mouse age; 10-week mice were sequenced to a minimum of 70 million reads/sample, while 26-week mice were sequenced to a minimum pf 40 million reads/sample.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

74907_hip

Data processing

Illumina bcl2fastq2 v2.20.0 software used for basecalling.
FASTQ files were trimmed, aligned to the mouse reference genome (GRCm38), sorted, and counted using the Bulk RNAseq Analysis Pipeline from the Minnesota Supercomputing Institute’s Collection of Hierarchical UMII/RIS Pipelines (v0.2.0 was used for 26-week-old data. v0.2.2 was used for the 10-week-old data). Component steps for read trimming, aligning and counting are as follows:
Reads were trimmed with Trimmomatic v.0.33 using illumina adapters, seedMismatches=4,palindromeClipThreshold=15, simpleClipThreshold=7, minAdapterLength=2 and keepBothReads=true
Trimmed reas were aligned to GRCm38 using hisat v.2.1.0 using the parameters --rna-strandedness RF and --no-mixed (suppress unpaired alignments for paired reads).
Aligned reads were assinged to genes using the featureCounts function of Subread v.1.6.2 with the parameters -B -p -Q10 -s2 and the Ensembl GRCm38 v.97 annotation gtf.
Assembly: GRCm38
Supplementary files format and content: subread_counts_10wk.txt is a matrix of gene counts for each sample at 10 weeks of age
Supplementary files format and content: subread_counts_26wk.txt is a matrix of gene counts for each sample at 26 weeks of age

Submission date

Nov 18, 2022

Last update date

Dec 01, 2022

Contact name

Christine M. Henzler

E-mail(s)

chenzler@umn.edu

Phone

6126257889

Organization name

University of Minnesota, Twin Cities (Minneapolis, MN, US)