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. 2012:786:181-200.
doi: 10.1007/978-1-61779-292-2_11.

CAGE (cap analysis of gene expression): a protocol for the detection of promoter and transcriptional networks

Hazuki Takahashi  1 , Sachi Kato, Mitsuyoshi Murata, Piero Carninci
Affiliations

CAGE (cap analysis of gene expression): a protocol for the detection of promoter and transcriptional networks

Hazuki Takahashi et al. Methods Mol Biol. 2012.

Abstract

We provide here a protocol for the preparation of cap-analysis gene expression (CAGE) libraries, which allows for measuring the expression of eukaryotic capped RNAs and simultaneously map the promoter regions. The presented protocol simplifies the previously published ones and moreover produces tags that are 27 nucleotides long, which facilitates mapping to the genome. The protocol takes less than 5 days to complete and presents a notable improvement compared to previously published versions.

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Figures

Fig. 1
Fig. 1. Length and shape of cDNA
cDNA quality check result of after Cap-trapping cDNA with Agilent RNA pico kit. One μl of the reaction was applied. Expected size ranges from few hundred nt to above 1-2 Kb. cDNA concentration is also measured by Oligreen (see Note 12 for the details).
Fig. 2
Fig. 2. Measurment of PCR products
PCR cycle check results with Agilent DNA 1000 kit. The amount of loaded PCR pro duct is 1 μl. (A) Result is shown for 13 cycles, (B) 15 cycles and (C) 18 cycles. In case of 13 cycles, the single peak (103 bp) has a FU value between 5 to 10 (molarity: ~10 nmol/l), which is usable for bulk PCR. In case of 15 cycles, the FU exceeds 20 (molarity: ~30 nmol/l) and with 18 cycles the reactions shows an additional broad peaks, due to overcycling. (D) Final product is measured with the Agilent DNA 1000 kit. The molarity of the single peak at 103 bp was estimated to be ~183.2 nmol/l. PCR primers are removed before the assay during the step 3.15, consisting of an ExoI treatment and Minelute column purification. The single peak products are ready for sequencing.
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