Hi experts,
I have a reporter construct that might have cryptic splice sites. I was wondering if it was possible to do direct RNA sequencing using Nanopore to get the distribution of transcript isoforms, but for this specific reporter mRNA.
I'm guessing you need to extract total RNA, enrich for polyA+ mRNA, and then somehow further enrich for your specific transcript? My reporter is transiently transfected, how much RNA would I need?
Thanks so much!
I don't know if you have considered this, but would you like to perform and amplicon based sequencing or direct sequencing? And I think that would decide the concentration you would be needing. On Illumina based targeted sequencing require "Low input (requires 10 ng of total RNA or 20 – 100 ng of FFPE RNA)" for amplicon sequencing.
https://emea.illumina.com/techniques/sequencing/rna-sequencing/targeted-rna-seq.html
Abhijeet Singh I had not considered! I'm a single molecule/microscopist so know very little about sequencing methods. Thanks for the link, their website has a lot of very useful information for me!
Hi Kelsey Bettridge
we did it in our lab mainly following this paper: Article Nanopore sequencing of full-length BRCA1 mRNA transcripts re...
somewhat easy (3'RACE PCR with the nanopore kits), we detected with right depth lot of transcripts (known and new) for our target.
all the best
fred