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Old 04-19-2013, 07:48 AM   #1
susma
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Default Is it how RNA-seq libraries should look like in Bioanalyzer?

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!
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File Type: pdf Pre-test RNA-seq library, #9, 12.04.2013.pdf (2.48 MB, 551 views)
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Old 05-01-2013, 12:09 AM   #2
Manal
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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.
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Old 05-01-2013, 11:12 PM   #3
susma
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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.
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Old 05-03-2013, 11:55 PM   #4
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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?
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Old 05-05-2013, 10:59 PM   #5
susma
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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.
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Old 05-05-2013, 11:02 PM   #6
Manal
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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.
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Old 05-06-2013, 12:04 AM   #7
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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
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File Type: pdf Pre-test 2 RNA seq library, #9, 15uM oligo, .pdf (1.85 MB, 159 views)
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Old 05-06-2013, 12:29 AM   #8
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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
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Old 05-06-2013, 11:10 PM   #9
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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.
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Old 05-07-2013, 07:14 AM   #10
susma
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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
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Old 05-21-2013, 04:42 AM   #11
Manal
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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.
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File Type: pdf Frag.pdf (305.4 KB, 186 views)
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Old 10-24-2013, 04:28 AM   #12
TonyBrooks
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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 ~5ÁL 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
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Old 10-24-2013, 05:18 AM   #13
Olaf Blue
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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-s....pdf?sfvrsn=10
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Old 03-21-2014, 08:51 AM   #14
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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?
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Old 03-22-2014, 04:06 AM   #15
susma
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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
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Old 03-24-2014, 01:46 AM   #16
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Hi Susma, that would be very helpful, look forward to your answers.
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Old 03-24-2014, 07:19 AM   #17
susma
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Hi again,

I checked my journal. It was rRNA contamination and I realized that the rRNA depletion kit using was not so efficient. I switched to Ribo-Zero kit and I got nice depletion afterwards and nice libraries.

If you want to make sure that you have the same problem or not, just run your RNA input on bioanalyzer. Then if you see the rRNA peaks (even small), it shows that you have the same problem.

Be careful that you are not overloading RNA when you are using Ribo-Zero kit for depletion. Otherwise, you will see the same spikes in your library traces.

I hope you get over this

Susma
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Old 03-24-2014, 08:35 AM   #18
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Many thanks for the information Susma, we were using old Turbo DNAse and RiboZero kits, so this may have resulted in less rRNA depletion, or maybe all DNA was not removed which can result in poor RiboZero performance. Anyway, good to know and we will check on Bioanalyzer for rRNA contamination.

Bruce.
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Old 05-15-2015, 02:24 PM   #19
ruoft
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Just FYI: My bioanalyzer traces looked exactly like this using the same kit. I ran the libraries on a gel and it pretty much replicated the bioanalyzer. Then I took two uL of each library and boiled it in 6uL FA dye to denature the sample and ran in on a 2% TBE gel. The upper bands disappeared and I got a nice smear from ~200-~600 bp. Maybe those spikes are the adaptor annealing products? (I did 14 cycles PCR and started with ~100ng RNA).
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