NanoCAGE-XL: An Approach to High-Confidence Transcription Start Site Sequencing
Identifying the transcription start sites (TSS) of genes is essential for characterizing promoter regions. Several protocols have been developed to capture the 5′ end of transcripts via Cap-Analysis of Gene Expression (CAGE) or linker-ligation strategies such as Paired-End Analysis of Transcription Start Sites (PEAT), but often require large amounts of tissue. More recently, nanoCAGE was developed for sequencing on the Illumina GAIIx to overcome this limitation. In this chapter, we present the nanoCAGE-XL protocol, the first publicly available adaptation of nanoCAGE for sequencing on recent ultra-high-throughput platforms such as Illumina HiSeq-2000. NanoCAGE-XL provides a method for precise transcription start site identification in large eukaryotic genomes, even in cases where input total RNA quantity is very limited.
Key wordsTranscription start site (TSS) Promoter NanoCAGE Capped analysis of gene expression High-throughput sequencing
We would like to thank Charles Plessy of the RIKEN Center for Life Science Technologies and Jenn To of Grassroots Biotechnology for technical advice on the nanoCAGE protocol. We would also like to thank Mark Dasenko of the Center for Genome Research and Biocomputing at Oregon State University for troubleshooting assistance in sample preparation for sequencing. This work was supported by NIH grant GM097188 and startup funds from Oregon State University to M.M.
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