Cap-analysis gene expression (CAGE) provides accurate high-throughput measurement of RNA expression. CAGE allows mapping of all the initiation sites of both capped coding and noncoding RNAs. In addition, transcriptional start sites within promoters are characterized at single-nucleotide resolution. The latter allows the regulatory inputs driving gene expression to be studied, which in turn enables the construction of transcriptional networks. Here we provide an optimized protocol for the construction of CAGE libraries on the basis of the preparation of 27-nt-long tags corresponding to initial bases at the 5′ ends of capped RNAs. We have optimized the methods using simple steps based on filtration, which altogether takes 4 d to complete. The CAGE tags can be readily sequenced with Illumina sequencers, and upon modification they are also amenable to sequencing using other platforms.
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- Supplementary Fig. 1 (652 KB)
Oligo-dT priming enhances the capture of CAGE tags on exons and 3′ UTRs.
CAGE libraries made from THP-1 cells. Data was displayed with the ZENBU genome browser (J. Severin, unpublished data). (a) The Actin beta gene is transcribed from right to left (violet arrow) on chromosome 7. (b) GAPDH gene is transcribed from left to right (green arrow) on chromosome 12. CAGE libraries were primed RT reaction with (1) random and oligodT (ratio 4:1) primers. (2) oligodT primers only and (3) random primers only. Both panels indicate that oligodT primers could enhance the capture of transcripts on 3′ exons and on internal exons, compared to random primer alone.