Hi,
I have made strand-specific RNA seq libraries. Some basic info is 500 ng total RNA (estimated from nanodrop) was inputted to a Turbo Dnase reaction, followed by Ribozero, fragmentation, first strand synthesis, dUTP strand-specific second strand synthesis and library prep, and 13 PCR cycles.
I realize the T16 sample is overamplified and that I need to do another clean-up to remove the adapter dimers, but what I wonder is if anybody has any insight specifically on is the jagged, or 'spiky', look of my final amplified libraries as assessed by HS DNA chip? I haven't seen this in my past libraries, although these past good libraris were made from 5 ug of different RNA and I modified my protocol a bit with this last batch.
I've also included intermediate BioA traces of the Ribozero'd RNA, fragmented RNA, and First Strand Synthesized RNA:cDNA hybrid.
My best guess is too low of input after the Dnase and various clean ups....
Thanks, K
I have made strand-specific RNA seq libraries. Some basic info is 500 ng total RNA (estimated from nanodrop) was inputted to a Turbo Dnase reaction, followed by Ribozero, fragmentation, first strand synthesis, dUTP strand-specific second strand synthesis and library prep, and 13 PCR cycles.
I realize the T16 sample is overamplified and that I need to do another clean-up to remove the adapter dimers, but what I wonder is if anybody has any insight specifically on is the jagged, or 'spiky', look of my final amplified libraries as assessed by HS DNA chip? I haven't seen this in my past libraries, although these past good libraris were made from 5 ug of different RNA and I modified my protocol a bit with this last batch.
I've also included intermediate BioA traces of the Ribozero'd RNA, fragmented RNA, and First Strand Synthesized RNA:cDNA hybrid.
My best guess is too low of input after the Dnase and various clean ups....
Thanks, K
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