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|Public on Oct 12, 2015
tissue: heart, biventricle
age: 17 weeks
|Hearts were removed and ventricles were flash frozen in liquid nitrogen. RNA was harvested using Trizol reagent.
Ribosomal RNA depletion along with RNA fragmentation and conversion to paired-end, strand-specific Illumina sequencing libraries were performed with Illumina TruSeq preparation kits for strand-specific, paired-end RNA-Seq.
|Illumina NextSeq 500
|rRNA-depleted total RNA
|Illumina Casava1.8 software was used for basecalling. All 76 nt reads use phred33 quality scores.
Sequence reads were mapped to the transcriptome, defined by annotation based on the UCSC mm10 genes.gtf but with mitochondrial RNAs added, and associated bowtie2 index provided by Illumina iGenomes (incorporating the additional mitochondrial RNAs), using tophat v2.0.10 (fr-firststrand)
htseq-count (authored by Simon Anders) was used to assign tophat-aligned reads to gene entries in the supplied gtf, based on UCSC mm10 genes.gtf but with mitochondrial RNAs added.
htseq-count data allows each user maximum flexibility in performing sample (library) depth normalization and differential gene expression using software such as DESeq or edgeR.
Additionally, we used Cufflinks v2.1 to calculate FPKM for each gene entry in each library. However, this was merely done for later graph display. Actual calculations of differential gene expression used htseq-count data as input to DESeq.
Supplementary_files_format_and_content: single tab-delimited text files include htseq-count values for each gene, or FPKM values for each gene.
|Oct 11, 2015
|Last update date
|May 15, 2019
|Scot J Matkovich
|Washington University School of Medicine
|Center for Cardiovascular Research
|660 S Euclid Ave, box 8086
|Chronic cardiac contractile dysfunction without hypertrophy does not provoke a compensatory transcriptional response