TY - JOUR T1 - NanoCAGE-XL: An Approach to High-Confidence Transcription Start Site Sequencing JF - Methods in Molecular Biology Y1 - 2018 A1 - Ivanchenko, Maria G A1 - Megraw, Molly AB -

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.

VL - 1830 UR - https://link.springer.com/protocol/10.1007/978-1-4939-8657-6_13 ER - TY - JOUR T1 - NanoCAGE-XL and CapFilter: an approach to genome wide identification of high confidence transcription start sites. JF - BMC Genomics Y1 - 2015 A1 - Cumbie, Jason S A1 - Ivanchenko, Maria G A1 - Megraw, Molly KW - Arabidopsis KW - Genes, Plant KW - Genome, Plant KW - Nanotechnology KW - Plant Roots KW - Promoter Regions, Genetic KW - Sequence Analysis, DNA KW - Software KW - Transcription Initiation Site AB -

BACKGROUND: 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 these difficulties.

RESULTS: Here we present the first publicly available adaptation of nanoCAGE for sequencing on recent ultra-high throughput platforms such as Illumina HiSeq-2000, and CapFilter, a computational pipeline that greatly increases confidence in TSS identification. We report excellent gene coverage, reproducibility, and precision in transcription start site discovery for samples from Arabidopsis thaliana roots.

CONCLUSION: nanoCAGE-XL together with CapFilter allows for genome wide identification of high confidence transcription start sites in large eukaryotic genomes.

[Link to Protocol, Additional Data, and Supplementary Materials]

[Link to CapFilter Software]

VL - 16 ER -