2 peaks in the library for RNA-seq

[circA]

Hi all,

I used KAPA RNA hyperprep kit to generate libraries and checked the distribution by Tapestation4200. However, there were 2 peaks in all my 6 samples(B1,C1,D1 and D2, E2, F2), one around 320nt, the other ~470nt.
I tried to heat and re-anneal these samples, however, after heating, it seemed that all peaks were gone(attached pic, lane E1, F1, G1,A2, B2,C2).

Is it OK to use these libraries for sequencing?


Another question is that I made a terrible mistake and heated the original libraries. Now all I have is ~8uL of diluted libraries. Can I amplify these libs again by PCR, because the RNA used for lib. preparation is quite limited?

Any suggestions are appreciated.
Thank you.

[nucacidhunter]

I would suggest to do 1-2 cycle PCR using all volume of denatured library as template and re-analyse. It is possible that libraries has not re-annealed so is not detectable by the Tape.

Amplification of diluted samples will distort the results.

[circA]

I would suggest to do 1-2 cycle PCR using all volume of denatured library as template and re-analyse. It is possible that libraries has not re-annealed so is not detectable by the Tape.

Amplification of diluted samples will distort the results.

Hi, thank you for your reply.
I did the reannealing by heating them to 95degrees for 2mins and then cooling to 45 degrees at 0.1deg/sec and then to RT by removing the heat block of the thermocycler.
Do you think this is OK for annealing?

I use 10mM tris(pH8.0) to store the libs and I think it should be OK to heat to 95 degs. However, it looks like all sequences are gone. If it is the reason you mentioned, do you think if it's possible to recover them by heating again and annealing again?

I did the quantification of the library by qPCR and the undiluted samples are quite concentrated(~100nM). Is it OK to use them as template and pcr for several cycles?

Thank you.

[nucacidhunter]

It should be enough for annealing low diversity short fragments such as adapter oligos. Denaturing should not destroy the DNA as in PCR this happens in every cycle. The fact that your fragments are not detected on Tape indicates that they have not been re-annealed. This is the reason that I suggested to do 1-2 PCR cycles.

Doing 2 cycles on majority of your library (denatured) will be better than several cycles of diluted library.

[pmiguel]

After heat denaturation you will probably still be able to visualize them on an RNA chip. Actually, I'm surprised the the tape station DNA fluor would be double-strand specific. The bioanalyzer DNA chip fluor isn't.

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Phillip