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Old 09-27-2021, 09:27 PM   #1
Chaos Indeterminate
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Location: India

Join Date: Sep 2021
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Default Strategy and Kits for mRNA and microRNA information from same sequencing

Hi!

I am from India and new to this forum.

I have no experience but still want to study differential mRNA and microRNA expressions from sorted human or mouse cells. I have access to a Illumina HiSeq sequencer. I want to figure out what kits and and reaction strategies would work best for the study.

Can anyone tell me whether paired end or unpaired and directional or unidirectional sequencing will work best for such experiments. If directional, should I go for 3' or 5' since I want both mRNA and microRNA from the same sequencing reaction and run.

Also to minimize cost, I want to get the mRNA and microRNA sequence and abundance from the same reaction. Is that a correct strategy?

Please advice on the correct strategy and kits, reads, depth of sequencing to enable maximum coverage for mouse and human.

Hope I could put my questions clearly.

I'm grateful for all help you all can extend and I need all of it

Thanks!!
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Old 09-29-2021, 10:50 AM   #2
luc
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Location: US

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To my knowledge, your idea is supported by only two kits, Perkin Elmer Combo-Seq and Bio-Rad SEQuoia. We have no experience with these, though.
I am not sure if the combined sequencing makes sense, though, due to the varying abundance of mRNAs and miRNAs and the presence of abundant other small RNAs in the sequencing data that will likely not be of interest. This would then require increased sequencing depth.
Commonly these two types of RNAs are sequenced separately with dedicated library prep protocols.
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illumina hiseq, kits, microrna sequencing, strategy

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