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Old 02-07-2017, 09:18 AM   #4
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Join Date: Jun 2012
Posts: 109

That thread specifically covers the SMART-Seq2 protocol, so there might be other considerations depending on what method you're planning on using. The recommendations there will be appropriate for template-switching based protocols, but possibly not homopolymer tailing (Tang, Quartz-Seq) or Eberwine (CEL-Seq(2)) based amplification. For example, SSIII has better thermostability and overall RT efficiency than SSII, but is bottlenecked by lower template-switching efficiency in protocols that need it, and some of the extra robustness in SSIV and Maxima appear necessary to overcome inhibitors in the newer bead based massive-multiplexing protocols but not solution based. Most of the protocols being published recently have included at least a little bit of their optimization path, which should could save a lot of time and cost if you were thinking of modifications. As a side note, I've wondered why there hasn't been more love for SMARTScribe. It works at least as well as SSII, is actually QC'd with a template-switching based assay, and is dirt cheap compared to other RNaseH- RTs (Full disclosure: I'm a former Clontech employee, but have since made single cell products for other companies and recently left the scRNA-Seq field but still lurk).
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