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Hello everyone,
I have a question about strand specific libraries (or lack thereof). I recently constructed total RNA-seq libraries using the V2 Script-seq kit, which is strand specific. Upon mapping my libraries, the data isn't looking strand specific. Many of the reads going the wrong way are spliced (meaning they cannot be a true biological phenomena) and they tend to occur at the 3' end of the transcript. I mapped with tophat2 followed by bowtie2 (using soft clipping). I have used this mapping protocol before and the strand specificity was pretty tight. The only thing I did differently with these new libraries is that I used Super Script IV in the RT step so I could sequence structured transcripts (SSIV can work at up to 60C). Does anyone have any ideas as to why I lost strand specificity? Could the SSIV be the cause? Thanks in advance.
I have a question about strand specific libraries (or lack thereof). I recently constructed total RNA-seq libraries using the V2 Script-seq kit, which is strand specific. Upon mapping my libraries, the data isn't looking strand specific. Many of the reads going the wrong way are spliced (meaning they cannot be a true biological phenomena) and they tend to occur at the 3' end of the transcript. I mapped with tophat2 followed by bowtie2 (using soft clipping). I have used this mapping protocol before and the strand specificity was pretty tight. The only thing I did differently with these new libraries is that I used Super Script IV in the RT step so I could sequence structured transcripts (SSIV can work at up to 60C). Does anyone have any ideas as to why I lost strand specificity? Could the SSIV be the cause? Thanks in advance.