title: Next-generation sequencing-based 5' rapid amplification of cDNA ends for alternative promoters.
abstract: Mammalian genomes contain many unknown alternative first exons and promoters. Thus, we have modified the existing 5'RACE (5' rapid amplification of cDNA ends) approach into a next-generation sequencing (NGS)-based new protocol that can identify these alternative promoters. This protocol has incorporated two main ideas: (i) 5'RACE starting from the known second exons of genes and (ii) NGS-based sequencing of the subsequent cDNA products. This protocol also provides a bioinformatics strategy that processes the sequence reads from NGS runs. This protocol has successfully identified several alternative promoters for an imprinted gene, PEG3. Overall, this NGS-based 5'RACE protocol is a sensitive and reliable method for detecting low-abundant transcripts and promoters.
authors.affiliation: Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA.
authors.forename: Bambarendage P U
authors.initials: BP
authors.lastname: Perera
authors.affiliation: Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA. Electronic address: jkim@lsu.edu.
authors.forename: Joomyeong
authors.initials: J
authors.lastname: Kim
mesh_terms: D000818:Animals; D018076:DNA, Complementary / Q000737:chemistry* / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000235:genetics; D051379:Mice; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA
chemical_list: D018076:DNA, Complementary; D051741:Kruppel-Like Transcription Factors; C490820:PEG3 protein, human; C097901:Peg3 protein, mouse
keywords: 5′RACE; Alternative promoters; NGS; Peg3