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achamess 02-18-2017 07:49 AM

Interpretation of Gene Body Distribution from nuclear RNA-Seq
Hi all,

So I just did an experiment using isolated RNA from neuronal nuclei. I used Smart-Seq2 to amplify from ~100 pg of input. I got plenty of cDNA that looked good on the Tapestation (single peak around ~1500 bp, spanning from 500 to 1000 bp). I then used the Nextera XT kit to finish the library prep. I ran as 50 bp SR on the HiSeq 4000.

I'm doing my QC stuff now after alignment with STAR to the mm10 Genome (UCSC). With respect to the gene body, I got interesting looking plots that I'm not entirely sure how to interpret. Some of the curves dip in the middle, and it looks like there is a slight 5' bias. The Nextera Kit looks funky in the first ~15 bp of the 5' end, but that's expected I've been told.

Any thoughts? Also, I'll say that I got this plot with RSeQC using a mm10 BED supplied on the site. I know that a large fraction (~40%) of my reads are intron, despite the polyA capture of Smart-Seq2. Could that be influencing the curves?

Thanks so much.

Here is the graph ->Graphs Gene Body Distribution Curves

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