I am preparing the library for single end RNA-seq following the illumina protocol. However, by agilent analysis for library, the two smear bands occur at 200bp and 500 bp. I do not know why the 500 bp band is visible. Who has the same experience? Anyone's idea for this result?
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What stage of the library prep are you at?? Is this the final purified library? What size bands are you selection? -
It is purified library for subsequent cluster generation in mRNA-seq. This library is generated by PCR enrichment during which the gel-seclection size at 200 bp is used as template. The PCR cycle is 15. Thus, in theory and according to illumina protocol of sample preparation for single end RNA-seq, the band shoud be only at around 200 bp, and the 500 bp band should not occur. I do not know why 500 bp band is yielded? I need 200 bp band.Originally posted by peromhc View PostComment
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What about running another gel and selecting only the size you want?Comment
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Poly, I would follow LMc's advice and do another round of size selection- it's hard to know what that band might be, but Im pretty sure you dont want it!Comment
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