RNA-seq analysis for off-targets induced by siRNA

Question

[M.Lee]

Hi, I am absolutely new to RNA-seq analysis. I would like to use this method to identify off-targets for my siRNA treatment. Basically i transfected or not transfected my cells in triplicate with that siRNA and then did RNA-seq in RNAs extracted from the samples for differential expression analysis (see how many genes were deferentially regulated). For the data generated, my colleague first aligned + mapped the library with STAR aligner, followed by the use of StringTie and Ballgown to study differential expression.

As an absolute rookie, my impression for RNA-seq is a heat map. However, in my case the transcriptome of treated cells is compared with those untreated, and that makes me think a one-lane heat map might look a bit odd. If I understand correctly, a better data presentation for my case may be: 1) a fold-change vs expression level (CPM) plot, or possibly, or 2) a volcano plot maybe. I am not sure if these types of plots can be drawn by Ballgown or any packages compatible with R (the only program available to me)? Can I used EdgeR or DEseq2 on data which have been processed by STAR and then StringTie?

Any help would be much appreciated! Lee

Answer 1

[Sean Davis]

You can use edgeR and DESeq2, yes. The place to start is with the vignettes/user manual for either of them. You may need to do some data manipulation to get your data into the appropriate objects for DESeq2 or edgeR, so you may have to spend some time (think about a couple of weeks) learning the basics of R. The best approach is to find a local expert who can help you with the details the first time.

[M.Lee]

Thank you very much Sean! Sorry I have been out of lab since I posted that. Will give DESeq2 a try as a local expert uses that :)