Here, you can interactively analyze and visualize RNA-seq data for all excitatory cell populations in the hippocampus at multiple levels of granularity. Analysis can be hypothesis driven by supplying predetermined lists of genes, or the dataset can be explored in a gene discovery-based fashion using enrichment and/or differential expression queries. To get started, select the population(s) of excitatory cells you are interested in analyzing, choose your desired level of analysis, and follow the prompts. Any figures or data generated during online analysis can easily be downloaded. Additionally, the full dataset of gene expression values can also be downloaded for offline analysis in other environments.
Thanks for checking out HippoSeq. We have put together an example exploratory workflow to analyze differences in gene expression in CA3 pyramidal cells.
To end the tour hit the 'x' at the top of any popup or the [esc] key.
This first box determines which populations are seen when viewing individual genes and heat maps. We'll select all populations to begin.
Note all populations have been selected. Each box is checked and the corresponding hippocampal region is filled.
Let's begin our analysis by examining transcriptional differences between dorsal and ventral CA3 populations.
We'll select dorsal CA3 pyramidal cells.
We'll compare to ventral CA3 pyramidal cells.
We'll select thresholding values to guide exploratory analysis.
We'll search for genes that are enriched at least 25 fold.
We'll require expressed genes to be at least 10 FPKM.
We'll also require genes to be differentially expressed.
We'll set a threshold of FDR=0.05 for differential expression.
We'll search for genes obeying the selected criteria and highlight them on a scatterplot.
After a short load time, the scatterplot for the two transcriptomes is shown, and highlighted in red are genes that meet the supplied search criteria.
Genes matching the supplied search criteria can be investigated more in-depth in a heat map.
Genes meeting the selected criteria are shown in the heatmap. Note that expression across all populations is shown.
Let's restrict the visualized populations to CA3 pyramidal cells only, which we used in the initial search.
Note now only dorsal and ventral CA3 pyramidal cell data will be shown.
Refresh the graph
Now only the CA3 pyramidal cell populations are shown. Note that the color in the heatmap is dominated by the highest expressing genes.
We'll set normalized expression to view each gene normalized to its maximum value.
We'll also view replicate reproducibility.
Let's refresh the graph again.
Note now we can see the enriched genes solely in the CA3 populations.
Let's investigate one of these genes, Hrh3, more closely.
Clicking on Hrh3 in the heatmap produces a barplot view of the individual gene.
We can always return to the previous heatmap by clicking on the appropriate tab.
Or the whole transcriptome analysis.
Thanks for joining us on this tour. Now, choose your cell populations and begin exploring gene expression in the hippocampus!