[Manfredi Carta]

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

[Treitli Sebastian]

Hi,

 

My personal experience (especially with SeraMag Beads) is that the binding capacity of the carboxylated beads in guanidine buffers is more reduced. When I was using SeraMag carboxylated beads to purify plasmid DNA according to the protocols described here (#5.1) I always got low amount of plasmid-DNA recovered compared to a control (like column method). The moment when I removed the guanidine buffer and used PEG buffers, the protocols started to work much better.

Now, the protocols.io link which you posted suggests that the beads are prepared in a PEG buffer, however, I think the guanidine salt causes some issues and this is why you have low recovery. Among things which I would try, would be to add more beads (make  the stock solution more concentrated, like use 2 mL of beads /50 mL of stock solution when you prepare the beads), or don’t use SeraMag carboxylated beads, but try to adapt a protocol with silica coated beads from here. Silica works better in guanidine buffers, and it might solve your issue.

 

Best regards,

Sebastian

[Author]

Posts