I take RNA-seq experiment by reagent I bought,not kit.
Then I first need to fragment RNA and take reverse transcription.But I can not get cDNA from this experiment by sequencing the ssDNA by Qubit. The the following procedures for RNA-seq failed.
I have tested the fragmentation time and reverse transcription time for this.And I also try to mix dNTP or primer or both with RNA when taking fragmentation before reverse transcription.
No matter what I do,I can not get cDNA .
The quality of RNA ,I also test by agrose gel.
I think it is a simple experiment.Does anyone know the keys of this? help me ! Thank you very much!
Hi Zhu
If your RIN No.>2. I find reducing the fragmentation time to 30 sec and increasing the amount of cleaning RNA beads to work. If you can see some smears in your gel, you should be able to generate cDNA unless your RNA IS OVER 98% degraded.
Hello
I agree with 100%
The article in link will be help .
Good Luck
http://www.cell.com/molecular-cell/pdf/S1097-2765(16)30182-4.pdf
@soloman Maina
thans for your recommendation. But I do not fragment RNA binding to beads. I just want to fragment RNA I isolate in solution with Tris.HCl,K+,Mg++. Have you ever test the efficiency of cDNA from fragmented RNA?
Just use my library prep method for deeply fragmented RNA:
Shishkin, Nature Methods, 2015
Van Nostrand et al., 2016, Nature Methods.
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