cDNA sequencing on IONTorrent PGM

Biomicrogen · 05-08-2013, 06:46 PM

Greetings all,

I will be doing some single cell transcriptomics soon. I am going to extract RNA from small numbers of bacterial cells (<10) and then circularize (after rRNA depletion) and subject this to MDA using phi29. The resulting cDNA should be able to be shunted into the MuSeek Library Prep kit workflow right? I mean, cDNA can be treated just like sheered gDNA can't it? I can't think of a reason I wouldn't get good reads doing it this way.

I would appreciate any insight you all might have!

Thanks,
Brandon @ASU

IonTorrent · 05-09-2013, 07:26 PM

Hi Brandon,

While there's not an official supported protocol to do that with MuSeek, you're absolutely correct - full length ds cDNA can generally be treated like gDNA for most library prep methods and I would suspect it will work fine. You might also consider checking in with the Thermo tech support team for further tips: ts.molbio@thermofisher.com

jonathanjacobs · 05-13-2013, 05:21 PM

Should work fine - but you'll loose strandedness information if you have any overlapping ORFs on opposite strands.

Proksima · 11-12-2013, 01:45 AM

We did library sequencing of cDNA using MuSeek. But cDNA have to be ds for transpose to fragment it.

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05-08-2013, 06:46 PM	#1
Biomicrogen Junior Member Location: Arizona Join Date: Aug 2012 Posts: 6	cDNA sequencing on IONTorrent PGM Greetings all, I will be doing some single cell transcriptomics soon. I am going to extract RNA from small numbers of bacterial cells (<10) and then circularize (after rRNA depletion) and subject this to MDA using phi29. The resulting cDNA should be able to be shunted into the MuSeek Library Prep kit workflow right? I mean, cDNA can be treated just like sheered gDNA can't it? I can't think of a reason I wouldn't get good reads doing it this way. I would appreciate any insight you all might have! Thanks, Brandon @ASU

05-09-2013, 07:26 PM	#2
IonTorrent Member Location: Guilford, CT and S.F., CA Join Date: Jan 2010 Posts: 64	Hi Brandon, While there's not an official supported protocol to do that with MuSeek, you're absolutely correct - full length ds cDNA can generally be treated like gDNA for most library prep methods and I would suspect it will work fine. You might also consider checking in with the Thermo tech support team for further tips: ts.molbio@thermofisher.com

05-13-2013, 05:21 PM	#3
jonathanjacobs Member Location: Rockville, MD Join Date: Apr 2011 Posts: 23	Should work fine - but you'll loose strandedness information if you have any overlapping ORFs on opposite strands.

11-12-2013, 01:45 AM	#4
Proksima Junior Member Location: Europe Join Date: Mar 2013 Posts: 3	We did library sequencing of cDNA using MuSeek. But cDNA have to be ds for transpose to fragment it.