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Hi,
I'm trying out the Total RNA Seq Kit V2 for small RNA seq on the PGM.
My template comes from exosomes and use more or less 5ng of small RNA (miRNA) enriched template (comes quite clean in Bioanalyzer RNA Pico and Nano and performes well in qRT-PCR). Although I follow the protocol suggested by the company, I get an enrichment in self ligated (no insert) adaptors in some samples (DNA 1000 kit in Bioanalyzer - smear analysis). I do overnight ligation at 16oC and amplify the cDNA library afterwards.
Is there a way to get rid of these products, or adjust the protocol to reduce them? I do get a good amount of miRNA inserts but the self ligated products are like 70% of the total yield. These are non-barcoded libraries.
Any tips are welcome.
Thanks!
I'm trying out the Total RNA Seq Kit V2 for small RNA seq on the PGM.
My template comes from exosomes and use more or less 5ng of small RNA (miRNA) enriched template (comes quite clean in Bioanalyzer RNA Pico and Nano and performes well in qRT-PCR). Although I follow the protocol suggested by the company, I get an enrichment in self ligated (no insert) adaptors in some samples (DNA 1000 kit in Bioanalyzer - smear analysis). I do overnight ligation at 16oC and amplify the cDNA library afterwards.
Is there a way to get rid of these products, or adjust the protocol to reduce them? I do get a good amount of miRNA inserts but the self ligated products are like 70% of the total yield. These are non-barcoded libraries.
Any tips are welcome.
Thanks!