Automated workflow for preparation of cDNA for cap analysis of gene expression on a single molecule sequencer.
Automated workflow for preparation of cDNA for cap analysis of gene expression on a single molecule sequencer.
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DOI：
10.1371/journal.pone.0030809
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Hayashizaki Y
中科院分区：
文献类型：
--
作者：
Itoh M;Kojima M;Nagao-Sato S;Saijo E;Lassmann T;Kanamori-Katayama M;Kaiho A;Lizio M;Kawaji H;Carninci P;Forrest AR;Hayashizaki Y
基因表达帽分析（CAGE）是一种5 '序列标签技术，用于全局确定基因组中的转录起始位点及其表达水平，最近已适用于HeliScope单分子测序仪。尽管CAGE协议有显著的简化，但到目前为止，它仍然是一个劳动密集型协议。在这项研究中，我们着手使该协议适应机器人工作流程，这将增加吞吐量并减少处理。我们在这里介绍的自动化CAGE cDNA制备系统可以在8天内制备96个“HeliScope就绪”CAGE cDNA文库，而不是手动操作员的6周。我们比较了在手动文库中使用相同RNA和多个自动化批次获得的结果以评估重现性。我们表明，测序是高度可重复的，与手工文库相比，生产率提高了8倍。CAGE cDNA自动制备系统可同时制备96个CAGE测序样品。最后，我们讨论了如何将该系统用于Illumina/SOLiD平台上的CAGE，RNA-seq和全长cDNA生成。
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. 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. 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.
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影响因子：
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作者：
Farias-Hesson E;Erikson J;Atkins A;Shen P;Davis RW;Scharfe C;Pourmand N
通讯作者：
Pourmand N
影响因子：
48
作者：
Cloonan, Nicole;Forrest, Alistair R. R.;Grimmond, Sean M.
通讯作者：
Grimmond, Sean M.
影响因子：
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影响因子：
48
作者：
Goren, Alon;Ozsolak, Fatih;Shoresh, Noam;Ku, Manching;Adli, Mazhar;Hart, Chris;Gymrek, Melissa;Zuk, Or;Regev, Aviv;Milos, Patrice M.;Bernstein, Bradley E.
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Bernstein, Bradley E.
影响因子：
12.3
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Lennon NJ;Lintner RE;Anderson S;Alvarez P;Barry A;Brockman W;Daza R;Erlich RL;Giannoukos G;Green L;Hollinger A;Hoover CA;Jaffe DB;Juhn F;McCarthy D;Perrin D;Ponchner K;Powers TL;Rizzolo K;Robbins D;Ryan E;Russ C;Sparrow T;Stalker J;Steelman S;Weiand M;Zimmer A;Henn MR;Nusbaum C;Nicol R
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Nicol R