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Old 06-08-2018, 09:12 AM   #260
Simone78
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Location: Bonn (Germany)

Join Date: Oct 2010
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Quote:
Originally Posted by cmbetts View Post
There's tons of low MW products there indicating massive RNA degradation, which you have in both your cell and purified RNA samples. Did you check the RIN of your pure RNA? If it's high, then you've got an RNase contamination problem, otherwise you need to look at your cell/RNA isolation procedure. I would not proceed to library prep and sequencing with those samples.
I agree, the RNA controls should be much better and give tons of cDNA, especially the 100 pg and 1 ng. Lots of degraded stuff or leftover primers in general. S2 and S7 are decent, though. S3 and S4 still have cDNA left, the others are all degraded. You might want to be more stringent with bead cleanup (0.8X is sufficient) and/or decrease the amount of oligos you are using. Which cells are these, by the way? That would determine the number of PCR cycles and help understanding whether itīs possible to get some good cDNA in general. Although Smart-seq2 is rather robust, not all cells give good results and you might need to play around with the lysis buffer.
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