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(Reposting my question from Nanopore Community)
Hi all,
I'm attempting to get a good structural picture of a human transcript and have a few questions regarding my approach using SQK-PCS111.
1) My transcript is very low abundance, so I'm designing sequence specific primers in an attempt to capture both sense and antisense transcripts, plus various structural isoforms that come both Poly Adenylated and Non-PA. My input will be refined total RNA (polyA RNA + rRNA depeleted Non-PolyA RNA recombined to 200ng with 100ng of each group). Is there a better ratio to use? Would it be prudent to do 190ng of NPA with only 10ngPolyA to reflect the more natural 5% of polyA mRNA? To be clear, the seq-specific primers will not be binding to d(A) tails.
2) The protocol implies that only one primer should be designed with the adapter and was wondering if anyone had any experience doing this pooled primer approach.
Primer 1: Exon 1, 3' of the antisense transcript
Primer 2: Exon 16, 3' of the sense transcript
Primer 3: Exon 5, 3' of the antisense transcript
Primer 4: Exon 9, 3' of the sense transcript
4) While Primer 1 (exon 1) and Primer 2 (Exon 16) are the 3' ends of their respective transcripts, will Primer 3 & 4 and the RT rxn be affected by the overhang created by the primer annealing in the middle of the transcript?
5) Knowing the directions of my read is important to our project, and if i've followed the chemistry diagram correctly, am I correct in assuming that (when starting from ssRNA) the transcript without adapters is representative of the original RNA molecule?
6) Can/Should Adaptive sampling be used with this seq-specifc approach?
Thanks in advance
Hi all,
I'm attempting to get a good structural picture of a human transcript and have a few questions regarding my approach using SQK-PCS111.
1) My transcript is very low abundance, so I'm designing sequence specific primers in an attempt to capture both sense and antisense transcripts, plus various structural isoforms that come both Poly Adenylated and Non-PA. My input will be refined total RNA (polyA RNA + rRNA depeleted Non-PolyA RNA recombined to 200ng with 100ng of each group). Is there a better ratio to use? Would it be prudent to do 190ng of NPA with only 10ngPolyA to reflect the more natural 5% of polyA mRNA? To be clear, the seq-specific primers will not be binding to d(A) tails.
2) The protocol implies that only one primer should be designed with the adapter and was wondering if anyone had any experience doing this pooled primer approach.
Primer 1: Exon 1, 3' of the antisense transcript
Primer 2: Exon 16, 3' of the sense transcript
Primer 3: Exon 5, 3' of the antisense transcript
Primer 4: Exon 9, 3' of the sense transcript
4) While Primer 1 (exon 1) and Primer 2 (Exon 16) are the 3' ends of their respective transcripts, will Primer 3 & 4 and the RT rxn be affected by the overhang created by the primer annealing in the middle of the transcript?
5) Knowing the directions of my read is important to our project, and if i've followed the chemistry diagram correctly, am I correct in assuming that (when starting from ssRNA) the transcript without adapters is representative of the original RNA molecule?
6) Can/Should Adaptive sampling be used with this seq-specifc approach?
Thanks in advance