Authors: Chelsea Herdman and Bradley Demarest
The Gene Expression Explorer app allows users to visualize differential expression results from RNA-seq datasets.
This Shiny app is supported by the B2B Consortium Grant (http://www.benchtobassinet.com) and hosted on http://b2b.hci.utah.edu/shiny/zebrafish_heart_timecourse/
Zebrafish fish hearts were mechanically separated from embyronic zebrafish at
five ages: 24, 36, 48, 60 and 72 hours post-fertilization (hpf). The procedure was
repeated 4 times for 24, 36 and 48hpf timepoints and 5 times for 60 and 72hpf timepoints
for a total of 22 biological samples.
RNA was purified from approximately n = 100 hearts per replicate per timepoint and the sample was split equally to produce a total RNA library (Illumina RiboZero Gold Library Kit) and a small RNA library (QIAseq miRNA Library Kit). All libraries for both RNA classes were multiplexed into two single RNA-Seq libraries and sequenced on an Illumina HiSeq 2500. The Total RNA sequencing library was sequenced paired-end over eight lanes and the Small RNA sequencing library was sequenced single-end over two lanes.
Data is publicly available at https://b2b.hci.utah.edu/gnomex/gnomexFlex.jsp?requestNumber=468R
Transcript abundances were quantified using kallisto (Bray et al. 2016) and genome build
GRCz11 release 99 (jan2020.archive.ensembl.org). The R package Tximport was used to give a bias-corrected gene-level abundance estimation (Soneson et al. 2015).
Summed estimated counts were rounded to the nearest integer in order to run DESeq2 (Love et al. 2014) using a negative binomial LRT model correcting for replicate (counts~ replicate + timepoint). This model tests in an anova-like way whether one timepoint differs from any other timepoint across the series.
Gene panel plots can be displayed using normalized counts or rlog values from DESeq2 or transcripts per million from kallisto. The light grey dot represents the mean expression value for that gene at each timepoint and the dark grey dots represent the value for each replicate. The vertical line at each timepoint depicts the range of the data and a line has been drawn to connect the mean at each timepoint to show the expression profile.
The heatmap displays z-scores (computed using DESeq2 normalized counts) for the selected gene list using a red-blue color scale.
Please email Chelsea Herdman firstname.lastname@example.org with any questions or issues