GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM6426683

Query DataSets for GSM6426683

Status

Public on Sep 07, 2023

Title

e13.5heart_Nos1ap_null_male_rep3

Sample type

SRA

Source name

heart

Organism

Mus musculus

Characteristics

tissue: heart
age: embryonic day 13.5
Sex: male
genotype: Nos1ap_null
strain: C57BL6/J

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated from e13.5 heart tissue using RNeasy Mini Kit following manufacturers’ recommendations (Qiagen, MD) that included the on-column DNase digestion using RNase-Free DNase set (Qiagen, MD). KAPA Stranded mRNA-Seq kit (KAPA Biosystems, MA) was used to generate indexed Illumina platform sequencing libraries. Briefly, polyA RNA was captured from 1µg total RNA using magnetic oligo-dT beads. After elution from the magnetic beads, polyA RNA was fragmented to generate inserts ranging in size from 100-200 bp, followed by random priming and reverse transcription to generate double-stranded cDNA. Next, after performing a 1.8× SPRI cleanup using AMPure XP beads (Agencourt, IN), dAMP was added to 3’-ends of the cDNA fragments.
Indexed 3’-dTMP Illumina TruSeq adapters were ligated to cDNA fragments. Ligated fragments were subsequently size selected using PEG/NaCl SPRI solution and underwent PCR amplification (12 cycles) to generate the sequencing libraries. After performing a 1× SPRI cleanup using AMPure XP beads (Agencourt, IN), a sample from each library was used to assess library fragment size distribution by electrophoresis using BioAnalyzer High Sensitivity DNA Assay (Agilent Technologies, CA) and to assess library concentration by qPCR using KAPA library quantification kit (KAPA Biosystems, MA). Equimolar amounts of libraries were pooled and sequenced on an Illumina HiSeq 2500 instrument using standard protocols for paired end 100 bp sequencing with a desired sequencing depth of ~30 million paired end reads per library.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Illumina RNA-seq paired-end read fastq files were quality checked using FASTQC (version: 0.11.5) (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and then processed using Trimmomatic (version: 0.36), for removing adapters and other Illumina-specific sequences from the reads, and, for performing a sliding-window based trimming of low quality bases from each read (ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:10:1:TRUE LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36). For estimating gene and isoform expression levels, we first extracted reference transcript sequences from the mouse genome (GRCm38, primary assembly) based on the GENCODE (http://www.gencodegenes.org/mouse_releases/current.html) primary assembly gene annotation (release M10) and built STAR aligner indices using the RSEM software package (version: 1.2.31). Trimmed paired-end reads from each sample were then aligned to the reference transcript sequences by calling the STAR aligner within RSEM and using alignment parameters from the ENCODE STAR-RSEM long RNA-seq pipeline (--outSAMunmapped Within --outFilterType BySJout --outSAMattributes NH HI AS NM MD --outFilterMultimapNmax 20 --outFilterMismatchNmax 999 --outFilterMismatchNoverLmax 0.04 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --sjdbScore 1 --quantMode TranscriptomeSAM). Gene and isoform expression levels were then estimated in each sample from these transcriptome alignments using RSEM keeping in mind the strandedness of the prepared RNA-seq libraries (--forward-prob 0.0). Gene-level read count data generated by RSEM was compared between wildtype and mutant mice to assess differential gene expression using the DESeq software (version: 1.24.0). Only those genes where the sum of read counts across the 10 samples was >1, were retained for differential gene expression analysis. Although, release M10 of the GENCODE primary assembly gene annotation has 48,526 genes, we limited differential gene expression comparison to only protein coding genes (22,098). To address multiple hypothesis testing, observed P-values were adjusted based on Benjamini-Hochberg FDR procedure.
Assembly: mm10
Supplementary files format and content: Text (.txt) file with differential gene expression analysis results using DESeq.

Submission date

Aug 01, 2022

Last update date

Sep 07, 2023

Contact name

Ashish Kapoor

Organization name

University of Texas Health Science Center at Houston

Street address

1825 Pressler St. Rm SRB 530C

City

Houston