Dear all
I am preparing to do PacBio ISO-Seq sequencing for total RNA of HeLa cells infected with 7.5Kb virus, I am interested in the Viral transcripts. My question is about library preparation and divided into two parts. Part 1 of the question: after isolating the polyadenylated transcripts, which step comes first, cDNA synthesis, or size selection? the second part of the question is about the size selection process, will the RNA go through a shearing process and then size selection, or the size will be selected based on RNA classes?
As I am interested only in the viral transcripts which is about 7.5Kb, thus size selection based on the classes will give me a stronger signal. While in the case of size selection based on fragments of sheared RNA will lower the signal, please correct me if I am wrong as I am not sure how to deal with the coverage if RNA has to be fragmented before selecting the size
Many thanks
I am preparing to do PacBio ISO-Seq sequencing for total RNA of HeLa cells infected with 7.5Kb virus, I am interested in the Viral transcripts. My question is about library preparation and divided into two parts. Part 1 of the question: after isolating the polyadenylated transcripts, which step comes first, cDNA synthesis, or size selection? the second part of the question is about the size selection process, will the RNA go through a shearing process and then size selection, or the size will be selected based on RNA classes?
As I am interested only in the viral transcripts which is about 7.5Kb, thus size selection based on the classes will give me a stronger signal. While in the case of size selection based on fragments of sheared RNA will lower the signal, please correct me if I am wrong as I am not sure how to deal with the coverage if RNA has to be fragmented before selecting the size
Many thanks
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