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-   -   Need high number of short cDNA fragments (http://seqanswers.com/forums/showthread.php?t=88627)

AndrewP 04-05-2019 08:00 AM

Need high number of short cDNA fragments
 
Hi all,

My application requires short cDNA fragments (up to 500 bp) with a defined 5' tag. I don't have the option to fragment RNA before reverse transcription using Mg and heat or the option to fragment afterwards via tagmentation or sonication. Does anyone have any ideas for the best way to accomplish this?

cmbetts 04-05-2019 02:41 PM

Take a page out of old school RACE (or Tang/Quartz scRNA-Seq for a more modern example). Add a homopolymer tail using TdT, perform second strand with a complementary primer also with a defined tag, and then amplify with primers complementary to the tags. If using very low inputs, you may need to use semi-suppressive PCR to keep down the artifacts.

AndrewP 04-05-2019 06:17 PM

Thanks for the advice, but I'm thinking more along the lines of causing premature termination in the reverse transcriptase. I realize it's the opposite request of what most users need, but the efficiency of all downstream steps drops exponentially with increasing cDNA length.

nucacidhunter 04-05-2019 11:58 PM

A general approach is to prime RT reaction with a random sequences (6-8nt) followed with a defined 5' overhang and then size select the cDNA. Implementing this will depend on downstream application.


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