RESUMO
Specialized RNA-seq methods are required to identify the 5' ends of transcripts, which are critical for studies of gene regulation, but these methods have not been systematically benchmarked. We directly compared six such methods, including the performance of five methods on a single human cellular RNA sample and a new spike-in RNA assay that helps circumvent challenges resulting from uncertainties in annotation and RNA processing. We found that the 'cap analysis of gene expression' (CAGE) method performed best for mRNA and that most of its unannotated peaks were supported by evidence from other genomic methods. We applied CAGE to eight brain-related samples and determined sample-specific transcription start site (TSS) usage, as well as a transcriptome-wide shift in TSS usage between fetal and adult brain.
Assuntos
RNA/química , Análise de Sequência de RNA/métodos , Sequência de Bases , Encéfalo , Células-Tronco Embrionárias , Biblioteca Gênica , Humanos , RNA/genética , RNA/metabolismoRESUMO
The original version of this paper contained an incorrect primer sequence. In the Methods subsection "Rampage libraries," the text for modification 3 stated that the reverse primer used for library indexing was 5'-CAAGCAGAAGACGGCATACGAGATXXXXXXXXGTGACTGGAGT-3'. The correct sequence of the oligonucleotide used is 5'-CAAGCAGAAGACGGCATACGAGATXXXXXXXXGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'. This error has been corrected in the PDF and HTML versions of the paper.