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Old 10-05-2017, 10:40 AM   #1
skannan4
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Location: Maryland

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Default Desiccation step in scRNA-seq protocol

Hi! My lab is trying to set up the SCRB-seq single cell protocol from Soumillon et al. (original preprint is here: https://www.biorxiv.org/content/bior...1/003236-1.pdf). One of the first steps before RT has you treat the cells with proteinase K and incubate at 50C, followed by an RNA desiccation step by removing the plate seal and incubating at 95C for 10 minutes (this both inactivates the proteinase K and reduces the sample volume, which at that point is ~7 ul). After that you do RT.

Currently in my hands I consistently find that the RNA is dramatically degraded during the desiccation step (have done both Bioanalyzer and Qubit, the former of which I can upload here if helpful). We have measured samples immediately after the 50C incubation and 95C incubation to verify. Our sequencing core tech expressed some trepidation as well about incubating RNA at 95C. Any thoughts or advice on what might be happening here?

We do both incubations in separate mini-incubators that we spray down with RNAZap beforehand.
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Old 06-19-2019, 04:49 PM   #2
jamesbaye
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Hi skannan,

Do you have any update on this?

I've been attempting to replicate the SCRBseq method and have observed some degradation of the sample (< 1 kb cDNA after first amplification). I haven't fully narrowed it down yet but suspect the lysis step.

Thanks,
James.
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Old 06-19-2019, 05:27 PM   #3
luc
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We have not tried this protocol. One question is: are your cells in a buffer very low in metals like Mg++ ? The protocol suggests a 1:500 dilution of Phusion HF buffer (presumably 1:500 of a 1x buffer). The higher the metal concentration, the more fragmentation will be induced.
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