why rG and not dG in the template switch oligos (TSO) in single cell library preps?

Simone78 · 03-14-2012, 01:18 AM

Hi all,
I am preparing single cell libraries for RNAseq using the SMARTer II kit (Clontech) and I was wondering whether there is a reason why they use rG in the template switch oligo (TSO).
They start from total RNA and do a regular RT using oligo-dT primers to make the first strand of cDNA. When the reverse transcriptase reaches the 5′ end of the mRNA template, it preferentially adds 2-5 cytosines. The template-switching oligo, which has 2-5 guanosines, anneals transiently, and reverse transcriptase then switches template and synthesizes the complementary strand. But why do they need 3 rG at the 3´-end of this oligo (which is otherwise a regular oligo with deoxynucleotide bases)?
It would be much cheaper and easier to use 3 dGs, but I guess the company already tried (and discarded) this option.
Thanks a lot!

zhengz · 03-14-2012, 10:40 AM

RNA-DNA binging is stronger than DNA-DNA. The rG TSO would anneal better than a dG one to the short stretch of 2-5 cytosines.

pmiguel · 03-14-2012, 11:56 AM

That is just what the reverse transcriptase in question prefers, right? I don't think how well the oligo binds comes into it.

--
Phillip

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
How to make HiSeq indexed paired-end library with homemade oligos?	ostrakon	Illumina/Solexa	6	03-16-2012 05:22 AM
Library for single cell RNA seq???	zianeffy	Sample Prep / Library Generation	2	07-05-2011 01:46 PM
SPRI-TE Illumina library preps	GW_OK	Sample Prep / Library Generation	5	08-23-2010 10:13 AM
Question of small quantity of cell or single cell sequencing	hxygz	Sample Prep / Library Generation	1	07-17-2009 12:48 AM
Homemade illumina oligos and library prep kit	elly	Illumina/Solexa	3	03-24-2009 09:05 AM

03-14-2012, 01:18 AM	#1
Simone78 Senior Member Location: Basel (Switzerland) Join Date: Oct 2010 Posts: 207	why rG and not dG in the template switch oligos (TSO) in single cell library preps? Hi all, I am preparing single cell libraries for RNAseq using the SMARTer II kit (Clontech) and I was wondering whether there is a reason why they use rG in the template switch oligo (TSO). They start from total RNA and do a regular RT using oligo-dT primers to make the first strand of cDNA. When the reverse transcriptase reaches the 5′ end of the mRNA template, it preferentially adds 2-5 cytosines. The template-switching oligo, which has 2-5 guanosines, anneals transiently, and reverse transcriptase then switches template and synthesizes the complementary strand. But why do they need 3 rG at the 3´-end of this oligo (which is otherwise a regular oligo with deoxynucleotide bases)? It would be much cheaper and easier to use 3 dGs, but I guess the company already tried (and discarded) this option. Thanks a lot!

03-14-2012, 10:40 AM	#2
zhengz Member Location: sweden Join Date: Aug 2010 Posts: 23	RNA-DNA binging is stronger than DNA-DNA. The rG TSO would anneal better than a dG one to the short stretch of 2-5 cytosines.

03-14-2012, 11:56 AM	#3
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	That is just what the reverse transcriptase in question prefers, right? I don't think how well the oligo binds comes into it. -- Phillip