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Old 11-29-2015, 12:56 PM   #169
Simone78
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Location: Basel (Switzerland)

Join Date: Oct 2010
Posts: 208
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Quote:
Originally Posted by amcg View Post
Hi all,
I'm hoping to use the Smart-seq2 protocol to make rna-seq libraries from small populations (200-500) of cells collected by FACS. In this case would you advise sorting them directly into the lysis buffer (triton-x and RNase inhibitor) described in the Nature Protocols paper, or would it be better to extract the RNA first and then proceed with the reverse transcription on purified RNA?

Many thanks!
Hi,
even if your cells have a lot of RNA I wouldn´t do it. This is only my opinion, others might think differently. Every time you transfer RNA before any amplification step you lose molecules. I would sort directly into the lysis buffer, but be aware that 0.2% Triton might not be sufficient for lysing so many cells.
Best,
Simone
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