Is it how RNA-seq libraries should look like in Bioanalyzer?

[susma]

Hi everybody,

It is my first time that I make DNA libraries for RNA sequencing. After using the ScriptSeq V2 library preparation kit I ended up with the attached results. I have run one sample with different concentrations on the High sensitivity DNA chip. Actually I do not have any idea if these peaks are OK or not.

I would really appreciate if someone gives me an advise or explanation, I am running out of time for this project!

[Manal]

Hi susma
I just had a look at your libraries, it seems that you had problem in the fragmentation of your RNA as you have small peaks within the range, I dont know how your protocol is but I am using RNAseq Illumina protocol and i had this problem in the beginning till i figured out where the problem comes from, in RNA library prep the step of fragmentation is important and critical , it should be done at the optimal time and temperature. check this step.

[susma]

Hi Manal,

Yes, is seems like that. The problem is that I do not have any idea how to change this part, because it is in the protocol and should be optimized. I will try to figure it out and let you know the results.

[Manal]

Hi Susma
In my case I had to change the incubation time ( in Illumina protocol, the fragmentation step should be done at 94C for 8min) but i have optimize it to 7:30min, and it made a big difference.
what is your protocol suggest and which protocol you are using?

[susma]

Hi Manal,

I am using the ScriptSeq V2 library preparation kit protocol. It says 85C, 5min. I contacted them and they said try 94C for 8min. I did, but still the same problem, bad fragmentation.
It got just a bit better.

[Manal]

Ok then try to incubate it for 7:30min at 94C, and see what will happen. and also for better practice as RNA is very sensitive work through the protocol and navigate from step to another while your tubes on ice to avoid any other unwanted reactions.

[susma]

Manal, do you think that 30 sec will make such a difference? I put my last result (94C, 8 min) here then you can compare it to the first one which was in 85 C, 5min (It is in the first attachment above). Sample 2ng has an error from bioanalyzer.

Susma

[Manal]

Actually i was having similar results, the 30sec will make a difference and also in in my case i had problems with some of the adapters , i dodnt know what are the problems but they simply didnt work.
making libraries seem straight forward but sometimes need optimization.
you had some results in your second try but not perfect, try to optimize the fragmentation time and see what will happen but for me it worked very nicely

good luck

[susma]

That is right. I am going to do it today with another mRNA sample and 7.30 min incubation time. I will let you know the results.

[susma]

Hi Manal,

I tried again to make libraries, but still the same problem with fragmentation step. I used 94C, 7.30 min. I also changed my mRNA sample to make sure that there is not something wrong with this particular sample.

I don't know what to do

[Manal]

Hi susma
sorry for late reply
check the file attached it might be useful, in our lab we are following Illumina protocols and have been provided with this when we had fragmentation problem.

[TonyBrooks]

If you elute your mRNA of dT beads directly into your first-strand / fragmentation buffer, make sure the beads are 100% dry first as any residual wash solution can screw up your fragmentation.
What I do is leave ~5µL of wash buffer in the tubes, spin down for 10 seconds and place on the magnet. Then go in with a small volume pipette (P10) and remove the residual wash buffer. Leave to dry for 5 minutes, then elute with the frag buffer

[Olaf Blue]

The ScriptSeq fragmentation protocol of 85C/5 minutes is certainly changeable and I have recommended using Illumina fragmentation conditions (94C/8 min) as an alternative. You can see what the "ideal" profiles are for SSV2 libraries on page 11, Appendix 4 of the product protocol:

http://www.epibio.com/docs/default-source/protocols/scriptseq-v2-rna-seq-library-preparation-kit.pdf?sfvrsn=10

[bruce01]

Hi all, sorry to drag up an old thread but I am seeing exactly this happening. Did anyone resolve this issue and get rid of the peaks?

[susma]

Hi Bruce01,

I could solve this problem and got good libraries and good sequencing results at the end. As it was long time ago and I am at home now, let me check my journals on Monday and write you a precise answer.I know that it was not about fragmentation and temperature and so on...

You will hear from me soon,

Best,
Susma