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© 2015 Elsevier Inc. 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.

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Analytical Biochemistry

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