Hello,
I'm new to this forum, however have used it as a guest for some time. In our lab we want to do paired end RNA-Seq on Illumina (which we have access to). I have made some libraries with the ScriptSeq mRNA protocol from Epicentre. Actually I have not enriched for mRNA, but removed rRNAs. So it will be whole-transcriptome.
The size distribution I get looks somewhat similar to the examples in the ScriptSeq protocol. That is I have a broad distribution from approx. 200-1500 bp (DNA high sensitivity chip on BioAnalyzer 2100).
Now I'm reading that it is best to size-select within a narrow range (let's say 350-450 bp) to get optimal bridge-amplification. If we consider that the fragmentation is unbiased (which it may not be), size-selection shouldn't be a concern for quantitative output from sequencing data or?
I'm not sure how to proceed. The plan is to "fragment" the library on agarose gel electrophoresis. So I can get distributions from e.g. 200-350, 350-450, 450-550 bp and so on. How do you guys do this? Or do you size-select at all? I believe that paired-end sequencing on RNA-seq for our pursoses would not make sense if some overlap between the reads is not present so they can be assembled in full ~2x150 bp.
Hope somebody can give me some inputs or considerations on this issue.
I'm new to this forum, however have used it as a guest for some time. In our lab we want to do paired end RNA-Seq on Illumina (which we have access to). I have made some libraries with the ScriptSeq mRNA protocol from Epicentre. Actually I have not enriched for mRNA, but removed rRNAs. So it will be whole-transcriptome.
The size distribution I get looks somewhat similar to the examples in the ScriptSeq protocol. That is I have a broad distribution from approx. 200-1500 bp (DNA high sensitivity chip on BioAnalyzer 2100).
Now I'm reading that it is best to size-select within a narrow range (let's say 350-450 bp) to get optimal bridge-amplification. If we consider that the fragmentation is unbiased (which it may not be), size-selection shouldn't be a concern for quantitative output from sequencing data or?
I'm not sure how to proceed. The plan is to "fragment" the library on agarose gel electrophoresis. So I can get distributions from e.g. 200-350, 350-450, 450-550 bp and so on. How do you guys do this? Or do you size-select at all? I believe that paired-end sequencing on RNA-seq for our pursoses would not make sense if some overlap between the reads is not present so they can be assembled in full ~2x150 bp.
Hope somebody can give me some inputs or considerations on this issue.
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