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Old 02-07-2019, 12:14 PM   #1
cerikson
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Location: mountian view

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Default Options for poorly fragmented library.

Hi all,
I've been working for some months to collect samples and build libraries for an absolute RNA-seq method, which is preferably PCR-free. I've sent my samples off to QC and I was sad to find that my fragments are much larger than expected. I was shooting for 200bp inserts, but my library fragments have ended up in the range of 600-900bp depending on the library. I was planning to use the HiSeq4000. Which according to the specs doesn't play well with fragments greater than 600bp fragments. And exclusion amplification is going to be very problematic with my libraries differing in size so much. So I was thinking to moving to the HiSeq2500 or Nextseq 500, as neither use ExAmp tech. And I think they handle larger fragments better? I do have enough material for one more library prep if it comes to that.

Also on the yield front. I think the SpriSelect beads got overdried. I've re-eluted and ddPCR on some of my samples, and it looks like I'm getting around 100 femtomoles per sample, and I'm pooling 24 samples. So I should be good to go.

My samples are RHCE in the attached bio-analyzer traces. Also, any clues what the 280bp peak is?

Method:
Twelve xenopus embryos were bead beated and snap frozen in trizol, then extracted. 1.5 ug of total RNA was treated with turbo DNAase and then precipitated in 5M LiCl and resupended in H2O. Libraries were then constructed with 1 ug total RNA, with RIN < 8, using the NEBNext Ultra II RNA kit(E7770). Libraries were enriched with Poly-A selection (NEB E7490) and prepped according to the manufacture's protocol, with the insert being 200bp. Adapters were ligated with a five-fold dilution of Truseq DNA Sgl index adapters. The protocol was stopped at the end of adapter ligation, and sent for QC.
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Old 02-07-2019, 06:16 PM   #2
luc
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Default

PCR-free Y-adapter libraries can migrate in funky ways and can hide adapter dimers. In short they are difficult to QC.
I would suggest a diagnostic PCR with a small aliquot of the libraries:
https://dnatech.genomecenter.ucdavis...ree-libraries/

It also has been suggested in this forum to run them after denaturation on an RNA bioanalyzer assay as an alternative.
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fragment bias, fragmentation problem

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