Questions about single-cell seq and drop-seq?

07 August 2021 0 7K Report

Hello!

We are trying to set up a single cell platform using the drop seq technology (Article Highly Parallel Genome-wide Expression Profiling of Individu...

). However we got some problems

Is it better, to prevent mRNA detached from the bead, to use SSC buffer @ 4 degree rather than room temperature?

Is it better to add Rnase inhibitors together with DTT in the bead phase to prevent RNA get decomposed?

Is it necessary, after reverse transcription, to use exonuclease I to break down unused Poly dT primer on the STAMP beads? Anyway unused Poly dT primer will not get amplify in the next PCR section, as it do not have TSO piece in 3' end

Being limited by oligonucleotide synthesis, I have to use 19 bp poly dT (Tm 44.9) instead of 30 bp in original study (Tm 55.2). Will this induce failure of mRNA capture? Will this cause RNA detachment in Reverse transcription using Superscript II (@42 ℃)? Normal reverse transcription use 12-18 bp poly dT with Superscript, yet will this be a problem in the pool of STAMP beads? If this is a problem, how about start reverse transcription @ 37℃ or 40℃ for 20min, then rise it to 42℃？ Will superscript II be completely block at lower temperature?

Thank you!

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