Preparation of Mammalian Nascent RNA for Long Read Sequencing
| Autor: | Karla M. Neugebauer, Kirsten A. Reimer |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
RNA Splicing
Computational biology Biology Article Cell Line 03 medical and health sciences 0302 clinical medicine Complementary DNA Animals Humans Molecular Biology Gene Library 030304 developmental biology Mammals 0303 health sciences Sequence Analysis RNA cDNA library High-Throughput Nucleotide Sequencing RNA Ribosomal RNA Amplicon Chromatin RNA splicing RNA extraction 030217 neurology & neurosurgery |
| Zdroj: | Curr Protoc Mol Biol |
| ISSN: | 1934-3647 1934-3639 |
| DOI: | 10.1002/cpmb.128 |
| Popis: | Long read sequencing technologies now allow high-quality sequencing of RNAs (or their cDNAs) that are hundreds to thousands of nucleotides long. Long read sequences of nascent RNA provide single-nucleotide-resolution information about co-transcriptional RNA processing events-e.g., splicing, folding, and base modifications. Here, we describe how to isolate nascent RNA from mammalian cells through subcellular fractionation of chromatin-associated RNA, as well as how to deplete poly(A)+ RNA and rRNA, and, finally, how to generate a full-length cDNA library for use on long read sequencing platforms. This approach allows for an understanding of coordinated splicing status across multi-intron transcripts by revealing patterns of splicing or other RNA processing events that cannot be gained from traditional short read RNA sequencing. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Subcellular fractionation Basic Protocol 2: Nascent RNA isolation and adapter ligation Basic Protocol 3: cDNA amplicon preparation. |
| Databáze: | OpenAIRE |
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