Hi everybody,
I'm new to SMRT sequencing, so I thought you guys might be able to give me some advice regarding a project we're currently planning. So thanks a lot already in advance!
Basically, we want to get an overview about the variation and abundance of splice isoforms for a single gene, similar to this publication:
http://www.pnas.org/content/early/2014/03/12/1403244111
We already have cDNA, prepared for Illumina RNA-seq, but with the Clontech SMARTer reagents, so we expect to have the full-length transcripts in there. The "major" isoform is around 4kbp of length.
From there, I would like to do a PCR using primers targeting the first and last exon of our gene. However, we have multiple samples (probably around 10), and since the throughput, looking only at a single gene, should be sufficient for multiplexing, I thought about adding barcodes at the 5' ends of both primers. I would use some of the barcodes proposed by Pacbio:
So I would get double-stranded cDNA with barcodes on both ends. But how to proceed from there? Does another size-selection step make sense? Can I go on immediately with the SMRTbell library prep?
And does anyone have experience with this "early start" issue, i.e. the polymerase might have passed the barcode already before the first signal?
Thanks again for any advice!
Best,
Chris
I'm new to SMRT sequencing, so I thought you guys might be able to give me some advice regarding a project we're currently planning. So thanks a lot already in advance!
Basically, we want to get an overview about the variation and abundance of splice isoforms for a single gene, similar to this publication:
http://www.pnas.org/content/early/2014/03/12/1403244111
We already have cDNA, prepared for Illumina RNA-seq, but with the Clontech SMARTer reagents, so we expect to have the full-length transcripts in there. The "major" isoform is around 4kbp of length.
From there, I would like to do a PCR using primers targeting the first and last exon of our gene. However, we have multiple samples (probably around 10), and since the throughput, looking only at a single gene, should be sufficient for multiplexing, I thought about adding barcodes at the 5' ends of both primers. I would use some of the barcodes proposed by Pacbio:
So I would get double-stranded cDNA with barcodes on both ends. But how to proceed from there? Does another size-selection step make sense? Can I go on immediately with the SMRTbell library prep?
And does anyone have experience with this "early start" issue, i.e. the polymerase might have passed the barcode already before the first signal?
Thanks again for any advice!
Best,
Chris