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Old 07-26-2021, 04:21 AM   #1
LJCatg
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Location: Cambridge

Join Date: Jul 2021
Posts: 1
Question mRNA library prep kit selection

Hello all,

I am a trainee scientist about to begin my research project which involves the setup of RNAseq in a clinical laboratory.

I am inexperienced in this regard and have a couple of questions I hope you may be able to assist with.

My experiment is a small scale bulk RNAseq on PAXgene extracted samples with the aim of interrogating some non-canonical splicing variants, I expect the samples will be of suitable quality that polyA selection will be viable rather than rRNA depletion techniques. Samples will be sequenced on a NovaSeq 6000 so there will be plenty of sequencing up for grabs.

Firstly, I've been having some difficulty in selecting the best kit for my experiment, particularly when it comes to choosing between the TruSeq Stranded mRNA kit and the Illumina Stranded mRNA kit.

As far as I can tell, the Illumina kit is newer and more efficient in most areas, including input requirements, hands on time, and multiplexing capabilities, but the majority of papers running RNAseq seem to use the TruSeq kit rather than the Illumina one. Should I stick to the tried and tested TruSeq kit, I will be sending my data for bioinformatics analysis to another laboratory who utilised the TruSeq kit when running their experiment, so would it be beneficial to keep this constant?

Or should I go for what seems to be the technically superior kit in the newer Illumina offering?

Secondly, I am unsure whether I need to utilise a Globin clearing kit alongside my mRNA library preparation kit, as I believe globin mRNA would make up a significant proportion of reads following sequencing. The literature of current RNAseq experiments seems to suggest that globin depletion is not necessary, and that these reads can be removed bioinformatically post-sequencing, but I struggle to see how a Globin clear kit wouldn't be beneficial in increasing the number of relevant reads and maximising the useful sequencing output towards relevant transcripts.

Is it because the inclusion of this extra protocol could result in a slightly more degraded library? Or because the number of reads required for analysis will be met regardless of whether the globin mRNA is removed?

Any assistance would be much appreciated,
Thanks in advance,
LJC
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Old 09-27-2021, 08:53 PM   #2
Chaos Indeterminate
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Location: India

Join Date: Sep 2021
Posts: 2
Post Differential expression of mRNAs and microRNAs on the Illumina HiSeq platform.

Hi,

I thought I will add my query as well here.

I am new to this group and have zero experience with sequencing. We want to find the differential expression of mRNAs and microRNAs on the illumina HiSeq platform.

Can anyone suggest the best protocol for library preparation that will enable to get realistic data for both mRNA and microRNA sequences and abundance from a single protocol that will minimize the expenditure.

Also this is mouse or human, so what I learnt so far unidirectional sequencing maybe sufficient and no need for paired ends right? Also when I say unidirectional do I always mean the 5' end or the 3' end because we want microRNAs also that ay be important. So which unidirectional protocol should we follow, 5' or 3'??

I hope the questions make sense. Please do reply and help.

Thanks!!
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