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Old 10-11-2011, 05:41 AM   #1
Tali7
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Location: United Kingdom

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Default Truseq Small RNA kit - losing library in the final cleanup?

Hi there

I've been using the Illumina Truseq small RNA kit, to little success. Starting off with 1ug of total RNA (all with RIN values of 10 I should add!), I took the samples through the whole library prep process, ran them on the polyacrylamide gel (see attached gel pic) and saw faint bands. Unfortunately, when I ran the finished, purified libraries my Bioanalyser results were very substandard (see attached .pdf). Most of them were almost flat lines!! Only one (sample #2) looked vaguely decent (this being the library with the brightest bands on the gel).

I was wondering whether anybody else has had similar problems with this? I have followed the protocol completely, and left the samples to elute for ~18 hours - the protocol says you can leave them in the shaker overnight, so I would expect higher yield if anything!

I also ran the libraries pre-PAGE on the Bioanalyser, but it is difficult to decipher the traces, as I had major daisy-chaining issues.

Thanks guys!
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File Type: jpg 20110822 miRNAseq 1-6.jpg (26.4 KB, 104 views)
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File Type: pdf libraries 1-11 and 13 manual integration.pdf (1,014.3 KB, 124 views)
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Old 10-11-2011, 11:59 AM   #2
pmiguel
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Not sure there is a problem here. If you have 2 nM or higher concentration library, you are ready to roll -- well, presuming most of the DNAs are amplicons. (Did you do an enrichment PCR? If so, how many cycles?)

How about using a high sensitivity DNA chip if you want to appreciate the full glory of your libraries? A DNA 1000 chip may just not be sensitive enough.

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Old 10-11-2011, 11:48 PM   #3
Tali7
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Well that's comforting to know! I did the optional ethanol precipitation at the end to concentrate the libraries, so they should be >10nM... but if you think that 2nM is an acceptable amount with which I can proceed to sequence.. then that's great!

Yes I did an enrichment PCR (the Illumina-recommended 11 cycles) - Illumina actually say that you can go up to 15 cycles if necessary for the kit, but I want to avoid any bias as much as possible.

I was hesitant to submit my libraries to be sequenced as one of the sequencing technologists here was making small RNA libraries with the same kit at the same time, and her Bioanalyser traces were much better than mine, so we figured I would have to repeat. If you don't think that's necessary, so much the better.

And now you mention it, I think our in-house sequencing facility has just purchased some High Sensitivity chips.. may give those a go.

Thanks a lot Phillip
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Old 10-12-2011, 03:26 AM   #4
pmiguel
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2 nM is the new starting concentration for v3 chemistry on a HiSeq or HiScanSQ. Of course your sequencing core may still want 10 nM, I don't know.

We generally also do qPCR to check libraries.

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