Miki Kojima

CAGE: cap analysis of gene expression.

1Laboratory for Genome Exploration Research Group, RIKEN Genomic Sciences Center (GSC), Yokohama Institute 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan. 2 Genome Science Laboratory, RIKEN, Wako main campus, 2-1 Hirosawa Wako, Saitama, 351-0198, Japan. 3 K.K. Dnaform, Tsukuba Branch, 3-1 Chuo 8-chome, Ami Machi, Inashiki Gun, Ibaraki, 300-0332, Japan. 4Present address: Vaxine Pty Ltd., Department of Diabetes and Endocrinology, Flinders Medical Centre, Bedford Park, Southern Australia 5042, Australia. Correspondence should be addressed to Y.H. (rgscerg@gsc.riken.jp), P.C. (rgscerg@gsc.riken.jp) or M.H. (matthias. harbers@dnaforminc.com).

Automated Workflow for Preparation of cDNA for Cap Analysis of Gene Expression on a Single Molecule Sequencer

Background Cap analysis of gene expression (CAGE) is a 5′ sequence tag technology to globally determine transcriptional starting sites in the genome and their expression levels and has most recently been adapted to the HeliScope single molecule sequencer. Despite significant simplifications in the CAGE protocol, it has until now been a labour intensive protocol. Methodology In this study we set out to adapt the protocol to a robotic workflow, which would increase throughput and reduce handling. The automated CAGE cDNA preparation system we present here can prepare 96 ‘HeliScope ready’ CAGE cDNA libraries in 8 days, as opposed to 6 weeks by a manual operator.We compare the results obtained using the same RNA in manual libraries and across multiple automation batches to assess reproducibility. Conclusions We show that the sequencing was highly reproducible and comparable to manual libraries with an 8 fold increase in productivity. The automated CAGE cDNA preparation system can prepare 96 CAGE sequencing samples simultaneously. Finally we discuss how the system could be used for CAGE on Illumina/SOLiD platforms, RNA-seq and full-length cDNA generation.

Telomerase Reverse Transcriptase Regulates microRNAs

MicroRNAs are small non-coding RNAs that inhibit the translation of target mRNAs. In humans, most microRNAs are transcribed by RNA polymerase II as long primary transcripts and processed by sequential cleavage of the two RNase III enzymes, DROSHA and DICER, into precursor and mature microRNAs, respectively. Although the fundamental functions of microRNAs in RNA silencing have been gradually uncovered, less is known about the regulatory mechanisms of microRNA expression. Here, we report that telomerase reverse transcriptase (TERT) extensively affects the expression levels of mature microRNAs. Deep sequencing-based screens of short RNA populations revealed that the suppression of TERT resulted in the downregulation of microRNAs expressed in THP-1 cells and HeLa cells. Primary and precursor microRNA levels were also reduced under the suppression of TERT. Similar results were obtained with the suppression of either BRG1 (also called SMARCA4) or nucleostemin, which are proteins interacting with TERT and functioning beyond telomeres. These results suggest that TERT regulates microRNAs at the very early phases in their biogenesis, presumably through non-telomerase mechanism(s).