Hi everyone,
I get some weird results when measuring my RNA-seq libraries on Bioanalyzer and now desperately need an advice...
The libraries look like a peak of expected length (300-400 or 400-500) and a higher peak (>1000 bp according to BA) (the image is attached).
I'm using TruSeq RNA v.2 kit, following the protocol (except for using qiagen columns instead of ampure beads and a size-selection step after PCR enrichment).
This is the first time that I'm making RNA-seq libraries but I have some experience with standard DNA libraries - and none of them looked like this. It was always a peak of expected length, sometimes with traces of adapter dimers at 120 bp - but never any peaks higher than expected.
What puzzles me the most is that I got this AFTER size selection. I'm pretty sure that I cut the band about 300-500 bp and certainly not 1500-2000 bp! So this should be something generated from my 300-500 bp fragments. What could it be? A single-stranded DNA? If so, will it affect the sequencing resuts? Does it make sense to try to run these libraries or it would be safer to redo them?
Thank you very much in advance!
I get some weird results when measuring my RNA-seq libraries on Bioanalyzer and now desperately need an advice...
The libraries look like a peak of expected length (300-400 or 400-500) and a higher peak (>1000 bp according to BA) (the image is attached).
I'm using TruSeq RNA v.2 kit, following the protocol (except for using qiagen columns instead of ampure beads and a size-selection step after PCR enrichment).
This is the first time that I'm making RNA-seq libraries but I have some experience with standard DNA libraries - and none of them looked like this. It was always a peak of expected length, sometimes with traces of adapter dimers at 120 bp - but never any peaks higher than expected.
What puzzles me the most is that I got this AFTER size selection. I'm pretty sure that I cut the band about 300-500 bp and certainly not 1500-2000 bp! So this should be something generated from my 300-500 bp fragments. What could it be? A single-stranded DNA? If so, will it affect the sequencing resuts? Does it make sense to try to run these libraries or it would be safer to redo them?
Thank you very much in advance!
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