cDNA cleanup from guanidine-HCl buffer

Hi everyone,

We are working on a single nucleus RNA-seq library prep method for prion disease samples. Our objective is to find a way to decontaminate prions while retaining high quality libraries. There are two methods to inactivate prions: O/N digestion with a high concentration of proteinase K and incubation in high-molar guanidine buffer for >2h, e.g. 4M Gdn-HCl or Gdn-SCN. It isn’t really clear from the literature how much Gdn salt is needed and how long the incubation should be, but it would be good to be on the safe side.

We want to use both PK and Gdn inactivation sequentially, to make sure that the prions are gone. I am using cDNA produced using the smartseq2 RT-PCR protocol for testing purposes. Later, we aim to produce libraries with the 10x Chromium or a well-sorting method such as mcSCRB-seq and then proceed with decontamination of pooled cDNA. I can incubate cDNA in PK O/N and purify it with AMPure beads without any significant loss of sample or cDNA degradation. However, the Gdn treatment is more tricky.

I have tried to purify cDNA from Gdn-HCl using the clean-up method described in gmcSCRB-seq (https://www.protocols.io/view/gmcscrb-seq-protocol-2aegabe). I mixed cDNA with lysis buffer components to obtain cDNA in 5M Gdn-HCl with Mercapto-EtOH and NEB Phusion buffer (essentially, I had lysis buffer as described in step 1, containing cDNA) and incubated O/N at RT. Then, I added clean-up bead mix (step 8) to my sample at a 1:2 sample:bead mix ratio, incubated for 5min and cleaned up on a magnet with 80% EtOH.

Overall, I think achieved about 20% recovery (see Tapestation profiles below). Have you got any suggestions on improving the yield or alternative approaches? I’m somewhat new to this field, so feel free to point out any issues you might notice.

Tapestation profiles

– non decontaminated input: https://ibb.co/ZmPTRx9

– decontaminated: https://ibb.co/Qmp4yyV