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Sample GSM7904534

Query DataSets for GSM7904534

Status

Public on Jan 15, 2024

Title

Colon, 24 Months, Rep1 [24M.N1]

Sample type

SRA

Source name

Colon

Organism

Mus musculus

Characteristics

tissue: Colon
strain: C57BL/6J/Ukj
Sex: Male
age: 24 Months
library batch: Prep 3

Growth protocol

All the mice were kept solely for aging in a controlled environment and health status.

Extracted molecule

polyA RNA

Extraction protocol

RNA was isolated using the QIAzol/chloroform extraction method.
RNA libraries were prepared using TruSeq RNA stranded kit (Illumina, San Diego, CA) with polyA enrichment according to the manufacturer’s instructions.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

24M.N1

Data processing

RNA was isolated using the QIAzol/chloroform extraction method. RNA quality was determined via an Agilent Bioanalyzer 2100 using the RNA 6000 nano kit (Agilent Technologies, Santa Clara, CA, USA). An RNA Integrity Number (RIN) value of greater than 7 was considered to be the minimum quality for sequencing.
RNA libraries were prepared using TruSeq RNA stranded kit (Illumina, San Diego, CA) with polyA enrichment according to the manufacturer’s instructions. All libraries were sequenced on an NovaSeq 6000 machine (Illumina, San Diego, CA, USA) with an average of 36 million paired-end reads (2x100 bp) at Competence Centre for Genomic Analysis (CCGA, Kiel, Germany) .
Illumina TruSeq adapter sequences were trimmed from forward and reverse reads using Cutadapt (2.8) with minimum sequence overlap of 3 bp, at most 10% mismatches allowed and minimum read length filter for 20 bp, as well as a two-color-chemistry aware, 3’-end quality trimming for a phred-score >= 25 (nextseq-trim=25).
An additional quality filter was applied with Prinseq lite (0.20.4) for at most 8 unknown nucleotides (‘N’) per read, an overall mean read quality of at least phred-score 15 and a minimum quality trim from both end of the read for a minimum phred-score of 12.
Filtered reads were then mapped against Mus musculus reference genome (GRCm38.p6, mm10) via Hisat2 (2.1.0) with RNA strandedness set to FR, employing non-deterministic random seeds and suppressing the mixed alignments of read pairs. Only primary alignments for each read were kept, via a filtering step (-F 256) with Samtools (1.9).
Gene counts were extracted using the tool featureCounts from the subread package (2.0.1) with reverse stranded information, while ignoring chimeric fragments and allowing only properly mapped read pairs with feature sizes in between 50 bp to 600 bp.
Assembly: GRCm38.p6 (mm10)
Supplementary files format and content: Tabular delimited matrix text files of read counts per GRCm38 gene (rows) and sample (columns).

Submission date

Nov 16, 2023

Last update date

Jan 15, 2024

Contact name

Lena Best

E-mail(s)

l.best@iem.uni-kiel.de

Organization name

Christian-Albrechts-University Kiel

Department

Institute of Experimental Medicine