silly question r.e. TruSeq stranded mRNA

sweetph3 · 03-21-2014, 12:58 PM

Hi. I'll be receiving some library preps from a collaborator next week that were made with TruSeq Stranded mRNA kit. We will run them for her on our MiSeq.

She keeps emphasizing "STRANDED" in her messages. I just want to check here to make sure there isn't some special way of running stranded mRNA libraries on the MiSeq. I haven't run any stranded libraries before. I'm assuming the difference will come in the analyses.

Please let me know if I need to do anything special for the run. I have looked at the protocol and it doesn't say anything about special running directions.

Thanks for taking the time to answer my silly newbie-sounding question!

kwaraska · 03-21-2014, 01:19 PM

Nothing specific to running them. It is all in the way the analysis is done.

The difference is that a stranded(also known as directional) protocol puts one adapter on one side and another adapter on the other side of the RNA-then runs an RT. In this way you know what direction a strand should be read based on where it starts.

The other way of making RNAseq libraries is converting the RNA to dbl stranded DNA, then constructing the library. Since the adapters are the same on both ends you don't know which direction to read.

She is probably emphasizing it because she doesn't know if you need to do anything different either!

sweetph3 · 03-21-2014, 01:20 PM

That's what I thought, thank you so much for your quick response!

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03-21-2014, 12:58 PM	#1
sweetph3 Member Location: New Jersey Join Date: Jun 2013 Posts: 22	silly question r.e. TruSeq stranded mRNA Hi. I'll be receiving some library preps from a collaborator next week that were made with TruSeq Stranded mRNA kit. We will run them for her on our MiSeq. She keeps emphasizing "STRANDED" in her messages. I just want to check here to make sure there isn't some special way of running stranded mRNA libraries on the MiSeq. I haven't run any stranded libraries before. I'm assuming the difference will come in the analyses. Please let me know if I need to do anything special for the run. I have looked at the protocol and it doesn't say anything about special running directions. Thanks for taking the time to answer my silly newbie-sounding question!

03-21-2014, 01:19 PM	#2
kwaraska Senior Member Location: Boston,MA Join Date: Nov 2008 Posts: 122	Nothing specific to running them. It is all in the way the analysis is done. The difference is that a stranded(also known as directional) protocol puts one adapter on one side and another adapter on the other side of the RNA-then runs an RT. In this way you know what direction a strand should be read based on where it starts. The other way of making RNAseq libraries is converting the RNA to dbl stranded DNA, then constructing the library. Since the adapters are the same on both ends you don't know which direction to read. She is probably emphasizing it because she doesn't know if you need to do anything different either!

03-21-2014, 01:20 PM	#3
sweetph3 Member Location: New Jersey Join Date: Jun 2013 Posts: 22	That's what I thought, thank you so much for your quick response!