Dataset Information

Automated workflow for preparation of cDNA for cap analysis of gene expression on a single molecule sequencer.

ABSTRACT: 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.

SUBMITTER: Itoh M

PROVIDER: S-EPMC3268765 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Automated workflow for preparation of cDNA for cap analysis of gene expression on a single molecule sequencer.

Itoh Masayoshi M Kojima Miki M Nagao-Sato Sayaka S Saijo Eri E Lassmann Timo T Kanamori-Katayama Mutsumi M Kaiho Ai A Lizio Marina M Kawaji Hideya H Carninci Piero P Forrest Alistair R R AR Hayashizaki Yoshihide Y

PloS one 20120130 1

<h4>Background</h4>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.<h4>Methodology</h4>In this study we set out to adapt the protocol to a robotic workflow, which would increase throughput and re ...[more]

PMID: 22303458

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Project description:The analysis of single cell proteomes has recently become a viable complement to transcript and genomics studies. Proteins are the main driver of cellular functionality and mRNA levels are often an unreliable proxy of such. Therefore, the global analysis of the proteome is essential to study cellular identities. Both multiplexed and label-free mass spectrometry-based approaches with single cell resolution have lately attributed surprising heterogeneity to believed homogenous cell populations. Even though specialized experimental designs and instrumentation have demonstrated remarkable advances, the efficient sample preparation of single cells still lacks behind. Here, we introduce the proteoCHIP, a universal option for single cell proteomics sample preparation at surprising sensitivity and throughput. The automated processing using a commercial system combining single cell isolation and picoliter dispensing, the cellenONE®, allows to reduce final sample volumes to low nanoliters submerged in a hexadecane layer simultaneously eliminating error prone manual sample handling and overcoming evaporation. With this specialized workflow we achieved around 1,000 protein groups per analytical run at remarkable reporter ion signal to noise while reducing or eliminating the carrier proteome. We identified close to 2,000 protein groups across 158 multiplexed single cells from two highly similar human cell types and clustered them based on their proteome. In-depth investigation of regulated proteins readily identified one of the main drivers for tumorigenicity in this cell type. Our workflow is compatible with all labeling reagents, can be easily adapted to custom workflows and is a viable option for label-free sample preparation. The specialized proteoCHIP design allows for the direct injection of label-free single cells via a standard autosampler resulting in the recovery of 30% more protein groups compared to samples transferred to PEG coated vials. We therefore are confident that our versatile, sensitive, and automated sample preparation workflow will be easily adoptable by non-specialized groups and will drive biological applications of single cell proteomics.

Dataset Information

Automated workflow for preparation of cDNA for cap analysis of gene expression on a single molecule sequencer.

Publications

Automated workflow for preparation of cDNA for cap analysis of gene expression on a single molecule sequencer.

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