Smart-seq2 lowest input?

kobeho24 · 05-25-2015, 10:21 PM

Hi All,
I tried to figure out what is the lowest input of Smart-seq2 on our hand. It doesn't work when the input of total RNA amount is 0.1pg or 1pg (serially diluted with RNase Free water), while products with >10pg input looks just fine in bioanalyzer. Have you guys ever tried such assays?

Best!

Simone78 · 06-04-2015, 07:19 AM

never tried with total RNA below 10 pg but I can tell you that even T or B cells work (and they have VERY little mRNA!), you just have to increase the number of cycles a bit (21-22). In that case, you might have also to decrease the amount of primers and block all the oligos with biotin at the 5´-end, to avoid getting just adaptor dimers.

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
SMART-seq2 and Nextera XT DNA sample preparation kit	wacguy	Sample Prep / Library Generation	9	02-09-2015 01:00 AM
SMART Concatemeres	sylvain	Sample Prep / Library Generation	3	08-21-2014 10:34 PM
First index always lowest percent reads	creeves	Illumina/Solexa	12	05-22-2013 06:35 AM

05-25-2015, 10:21 PM	#1
kobeho24 Member Location: HKUST, Hong Kong Join Date: Apr 2015 Posts: 32	Smart-seq2 lowest input? Hi All, I tried to figure out what is the lowest input of Smart-seq2 on our hand. It doesn't work when the input of total RNA amount is 0.1pg or 1pg (serially diluted with RNase Free water), while products with >10pg input looks just fine in bioanalyzer. Have you guys ever tried such assays? Best!

06-04-2015, 07:19 AM	#2
Simone78 Senior Member Location: Bonn (Germany) Join Date: Oct 2010 Posts: 180	never tried with total RNA below 10 pg but I can tell you that even T or B cells work (and they have VERY little mRNA!), you just have to increase the number of cycles a bit (21-22). In that case, you might have also to decrease the amount of primers and block all the oligos with biotin at the 5´-end, to avoid getting just adaptor dimers.