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

Masayoshi Itoh, Miki Kojima, Sayaka Nagao-Sato, Eri Saijo, Timo Lassmann, Mutsumi Kanamori-Katayama, Ai Kaiho, Marina Lizio, Hideya Kawaji, Piero Carninci, Alistair R. R. Forrest, Yoshihide Hayashizaki (+1 others)
2012 PLoS ONE  
Cap analysis of gene expression (CAGE) is a 59 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
more » ... ted 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 fulllength cDNA generation.
doi:10.1371/journal.pone.0030809 pmid:22303458 pmcid:PMC3268765 fatcat:a2hevyytynck5py2ij25jfblqm