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Old 11-25-2015, 05:49 PM   #166
kobeho24
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Location: HKUST, Hong Kong

Join Date: Apr 2015
Posts: 32
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Quote:
Originally Posted by Simone78 View Post
Hi Gary,
I just tried with 0.2% SDS, in the same way as I am doing after tagmention to strip the Tn5 off the DNA before PCR and it didnīt work. I also tried SDS + NaOH/HCl (skipping columns or precipitation, as they recommend in the protocol) but, as I said, there was probably too much salt at that point and the PCR didnīt work.
/Simone
Hi Simone,
I don't know if you noticed that the newly published TGIRT paper in the journal RNA. They use the TGIRT to do the RT of human plasma RNA. And I realized that there is a fragmentation step prior to RT when dealing with whole cell total RNA as they suggested. Thus, on my perspective, defeat its better processivity compared to retroviral RT. You mentioned that the average size of cDNA generated by TGIRT is 4-5k. I just wonder if you have an in-house protocol to do so. It would be much appreciated if you let me know.
BTW, I noticed that the TGIRT can template switch not only RNA but also DNA, which means it might not be compatible with single-cell RNA-seq, unless we extract the total RNA from a single cell and do rRNA-depletion before RT, am I right?

Best!
Gary
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