I am preparing RNA samples for Illumina sequencing with the RT-Klenow protocol but I don't have reproducible results. Sometimes i get an important number of reads after sequencing, sometimes not and recently all the reads i get are millions and millions of only A and T bases.

I am not sure whether the problem comes from me or the NGS platform (after beads purification I give my samples to another platform where they generate the library, do the amplification etc).

So, I was thinking of adding an RNAase H step after RT and an Mung Bean nuclease+qPCR after Klenow. Someone also told me that random hexamers have to be in excess for the RT to work properly.

What do you think? Have you encountered the same problem?

Thank you in advance!