BIOO Scientific stranded RNAseq library prep

[teofil]

Dear members,

We are using the BIOO Scientific NextFlex rapid directional RNAseq kit to prepare RNAseq libraries and getting some unexpected results. Namely, in about half of our preps, TapeStation electropherograms show a marked peak at the low end of the library followed by a ~normal looking profile toward the higher end of the library (see attached pdf). In addition, and compared to nondirectional TruSeq preps routinely performed in the lab, we are getting quite low library yields (commonly ~ 1ng/uL and sometimes below that). The input RNA is 200 ng with RIN > 7. The RIN numbers are not great due to peaks from organelle rRNA (we think primarily chloroplast).

We suspect that we might be doing something wrong, but we are unsure what. Any suggestions would be great (fragmentation, PCR, Ampure cleanups).

Also, has anyone had experience sequencing libraries looking like this? We're planning to sequence on a HiSeq 2000.

Thanks!

[nucacidhunter]

I wonder if you have asked Bioo techsupport for troubleshooting. Technically those libraries can be sequenced, but they are less likely to provide publication quality data.