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Old 02-13-2019, 07:59 AM   #1
Lechu
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Location: Germany

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Default barcoded RNA adapter ligation for multiplexing

Hi All,

This is my first post here, so hello everyone!

I'm wondering if it is possible to use RNA adapter ligated to (preferably) 3' end of the RNA for multiplexing the samples prior to RNA sequencing? If I understand correctly (an my knowledge may be narrow here), typically such adapters are added separately to individual samples, and then the barcodes are added later by PCR.

I would like to:

Ligate RNA adapters into multiple samples, and then pool them together before performing cDNA synthesis. After ligation, samples would be multiplexed, and processed as such from the very beginning. Specifically, I would like to perform a 3'UTR (or its polyA adjacent part) sequencing, using the custom adapter-specific primer containing barcode. My cDNA library would be made as follows, (with optional polyA enrichment):

........................................................................................... <-----------
....NNNNNNNNNNNNNN-AAAAAAAA [...] AAAAAA-BARCODE-ADAPTER

I would then use a random primer and adapter-specific primer for generating a second cDNA strand, and add adapters for amplification on the flow cell by PCR.

I can imagine such approach might lead to biases in case the RNA concentrations and/or quality in individual samples is highly variable. But assuming it is not, I see it as a sound approach. Or am I missing something?

Thank is advance for any helpful insights.

Cheers,
Lech

Last edited by Lechu; 02-13-2019 at 08:09 AM. Reason: layout problems
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Old 02-14-2019, 12:52 PM   #2
cmbetts
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Default

Very doable, although random priming isn't necessary for second strand if you're using standard Gubler-Hoffman, the RNaseH handles the priming. This Nature Methods paper from the Broad should give some inspiration on how to do it https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4712044/
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