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.


METHODS
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.
Omics Science Center, RIKEN Yokohama Institute, Tsurumi-ku, Yokohama, Kanagawa, Japan. maitoh@riken.jp
Masayoshi
M
Itoh
Miki
M
Kojima
Sayaka
S
Nagao-Sato
Eri
E
Saijo
Timo
T
Lassmann
Mutsumi
M
Kanamori-Katayama
Ai
A
Kaiho
Marina
M
Lizio
Hideya
H
Kawaji
Piero
P
Carninci
Alistair R R
AR
Forrest
Yoshihide
Y
Hayashizaki
D000818:Animals; D001331:Automation; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D015723:Gene Library; D015894:Genome, Human / Q000235:genetics; D006801:Humans; D051379:Mice; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000295:instrumentation* / Q000379:methods*; D057188:Workflow
D018076:DNA, Complementary