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-   -   Reverse Transcriptase of choice for single cell cDNA synthesis (http://seqanswers.com/forums/showthread.php?t=74051)

microliter 02-03-2017 07:21 PM

Reverse Transcriptase of choice for single cell cDNA synthesis
 
Superscript III, Maxima H-?

Its difficult to parse out the differences between many of the commercially-available M-MuLV RTs. Does anyone have opinions or thoughts on their preferred enzymes?

huguesparri 02-06-2017 03:43 AM

You can take a look at this post:
http://seqanswers.com/forums/showthread.php?t=9967
It might be helpfull.

microliter 02-06-2017 01:42 PM

perfect. thank you

cmbetts 02-07-2017 09:18 AM

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).

plyguy69 02-15-2017 11:16 AM

I've used both. And ClonTech's RTs. I've personally found that Maxima and Primescript both work well at 50C and 42C respectively. Try the Primescript with Betaine and DTT in the mix and see if that works for you.
Cheers


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