Dear All
I used unstranded RNA-seq for non-coding RNA prediction, in 1st step I removed all sequence which is overlapping (in sense) exons of the reference annotation. so can I consider the remaining reads that map to exonic antisense strand as antisense transcript?
Thanks
Figure 6 of Griffith et al., 2015 offers a good illustration of ATpoint's explanation.
Yes, you have mapping to both strands but this is because adapter ligation is random and after PCR amplification, the strand-of-origin cannot be identified anymore unlike in stranded preps. Sorry about that but you cannot answer the question you have with this experimental design.
There are protocols for stranded qPCR. Still, you might consider preparing a new RNA-seq stranded library. Most sequencing facilities offer all-in service for library prep and sequencing for an affordable price. qPCR can be annoying and in the end only gives you information about a few regions.
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Tough. You can infer by going after annotated antisense transcripts and look for exonic regions that aren't overlapped with protein-coding transcripts in the sense direction. Then inspect the quants case by case and design primers to validate the findings experimentally. There's only so much you can accomplish this with unstranded protocols.