Long read transcript profiling of ion channel splice isoforms.

Hall NAL., Husain SM., Lee H., Tunbridge EM.

Alternative splicing of RNA transcripts allows a single gene to generate multiple products and is a key means of generating functionally diverse voltage-gated ion channels. Splicing can be regulated according to cell type, cell state, and stage of development to produce a bespoke complement of protein isoforms. Characterizing the identities of full-length transcript isoforms is essential in order to fully understand a gene's expression and function. However, the repertoire of transcript isoforms is not well characterized for most genes. Long read nanopore sequencing allows full-length isoforms to be sequenced, therefore identifying full-length transcripts. Using this approach, we recently discovered that the human CACNA1C gene gives rise to a far greater repertoire of splice isoforms than previously appreciated. Here we provide a detailed overview of the technical approach we used to achieve this. The method described in this chapter combines long read nanopore sequencing with PCR targeting to selectively sequence transcripts of a specific gene of interest.

DOI

10.1016/bs.mie.2021.02.015

Type

Chapter

Publication Date

2021-01-01T00:00:00+00:00

Volume

654

Pages

345 - 364

Total pages

19

Keywords

Alternative splicing, CACNA1C, Calcium channel, Gene expression, mRNA, Alternative Splicing, Gene Expression Profiling, Humans, Ion Channels, Protein Isoforms, RNA Splicing

Permalink More information Close