I find myself in a predicament that I'm not sure how to resolve:

I have managed to isolate a particular multi-cellular structure from postmortem human brain tissue with the intention of isolating RNA from that structure and building libraries for RNA-sequencing.

Reagents and kits used:

Single Cell RNA Purification Kit from Norgen

RNase-Free DNase I Kit from Norgen (on column DNase treatment)

SMARTer Stranded Total RNA-Seq Kit v3 - Pico Input Mammalian library kit from Takara

So far, RNA extraction from that structure has been successful and so has library building with one issue: There is a second, consistently present, smaller peak at a larger size than a library. So I either have the recurring issue of either gDNA (despite performing DNAse treatment) and/or over-amplified products in library traces. No matter how I have tried to adjust RNA input and PCR cycle, a second peak keeps cropping up in library traces.

I think I’ve managed to reduce the size of the second peak as much as I can to the extent where I don’t think my libraries are over-amplified and whatever it is, is too large from my library to be sequenced (see attached file Takara V3 Library Kit Optimization Conditions 6, specifically well B1). Would such a library be adequate for sequencing? I have received the criticism that my desired library peak may also have gDNA - how likely is this, given the shape of the trace? I know some genomic contamination is inevitable but I'm hoping to keep it as low as possible.

Side note for those who don't work with postmortem brain: lower RINe, lower yields in general, and lower quality "everything" is to be expected. So I’m also concerned that lowering amplification more will not be sufficient for a number of lower quality samples.

Any advice would be greatly appreciated!