NanoCAGE: A Method for the Analysis of Coding and Noncoding 5′-Capped Transcriptomes
Transcripts in all eukaryotes are characterized by the 5′-end specific cap structure in mRNAs. Cap Analysis Gene Expression or CAGE makes use of these caps to specifically obtain cDNA fragments from the 5′-end of RNA and sequences those at high throughput for transcript identification and genome-wide mapping of transcription start sites for coding and noncoding genes. Here, we provide an improved version of our nanoCAGE protocol that has been developed for preparing CAGE libraries from as little as 50 ng of total RNA within three standard working days. Key steps in library preparation have been improved over our previously published protocol to obtain libraries having a good 5′-end selection and a more equal size distribution for higher sequencing efficiency on Illumina MiSeq and HiSeq sequencers. We recommend nanoCAGE as the method of choice for transcriptome profiling projects even from limited amounts of RNA, and as the best approach for genome-wide mapping of transcription start sites within promoter regions.
Key wordsCap analysis gene expression CAGE nanoCAGE CAGEscan RNA Transcription Start Sites TSS Expression profiling Template switching Tagmentation Multiplexing Unique molecular identifiers UMI
We thank Alexandre Fort for critically reading the manuscript and his helpful comments, and Laia Masvidal Sanz for helpful discussions and suggestions on the nanoCAGE protocol. This work was founded by a Research Grant from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) to the RIKEN Center for Life Science Technologies, and a JSPS Grant-in-Aid for Young Scientists A (number 25710018).
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