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Old 09-13-2016, 05:28 PM   #1
Fitzcarraldo
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Default Recommended kit for RNAseq transcriptomic analysis of lncRNAs

We have extracted total RNA from virus infected and uninfected human cell lines at different time points in triplicate and purified with Trizol and Direct-zol RNA prep (Zymo Research with DNase I on-column treatments).

The RIN values are all >8 which is lower than the >9.5 seen with non-DNase treated samples although seems to be a known effect.
http://seqanswers.com/forums/showthread.php?t=20699

Can I please ask which kits are recommended to prepare a library for HiSeq analysis of lncRNAs?

We have the Illumina TruSeq Stranded mRNA Library Prep kit and we are interested in host transcriptomic responses, particularly those of long non-coding RNAs (lncRNAs).

My question is can this Illumina kit be employed for transcriptomic analysis including that of host mRNA and lncRNAs differentially regulated upon infection?

As the majority of host lncRNAs are polyadenylated I think this should be possible and would like to confirm. Illumina's response was non-committal and said they had not validated the kit for this purpose. There are a fraction of lncRNAs not polyadenylated (like rRNAs which we would like to remove) which is concerning me as is the decreased RINs after DNase treatment but we considered removal of the gDNA to be necessary.

Many thanks in advance for your time and expertise and would be deeply grateful for any advice or comments.

Best wishes
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Old 09-13-2016, 08:34 PM   #2
luc
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To my knowledge only about 90% of the lncRNAs tend to be polyadenylated. Most lncRNA studies I have seen use poly-A enrichment nevertheless.
The Illumina kit should be as good as any other kit (BiooScientific, Nugen, Kapa, NEB, Agilent, ...).

We always do the DNAse treatment off-column using the same Zymo kit; we have not noticed an impact on RIN scores.
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Old 09-13-2016, 09:48 PM   #3
nucacidhunter
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The best option is one of Stranded Total RNA-Seq kits which uses rRNA depleted RNA as input. These type of kits costs more and also require more sequencing. In return you might be able to discover new transcripts that have been overlooked using mRNA approaches. I remember that once there was a one gene, one protein hypothesis and majority of scientists believed it.
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