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Four methods of preparing mRNA 5' end libraries using the Illumina sequencing platform.

ABSTRACT: BACKGROUND: The 5' untranslated regions of mRNA play an important role in their translation. RESULTS: Here, we describe the development of four methods of profiling mRNA 5' ends using the Illumina sequencing platform; the first method utilizes SMART (Switching Mechanism At 5' end of RNA Transcript) technology, while the second involves replacing the 5' cap structure with RNA oligomers via ligation. The third and fourth methods are modifications of SMART, and involve enriching mRNA molecules with (nuclear transcripts) and without (mitochondrial transcripts) 5' end cap structures, respectively. Libraries prepared using SMART technology gave more reproducible results, but the ligation method was advantageous in that it only sequenced mRNAs with a cap structure at the 5' end. CONCLUSIONS: These methods are suitable for global mapping of mRNA 5' ends, both with and without cap structures, at a single molecule resolution. In addition, comparison of the present results obtained using different methods revealed the presence of abundant messenger RNAs without a cap structure.

SUBMITTER: Machida RJ

PROVIDER: S-EPMC4086933 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Four methods of preparing mRNA 5' end libraries using the Illumina sequencing platform.

Machida Ryuji J RJ Lin Ya-Ying YY

PloS one 20140708 7

<h4>Background</h4>The 5' untranslated regions of mRNA play an important role in their translation.<h4>Results</h4>Here, we describe the development of four methods of profiling mRNA 5' ends using the Illumina sequencing platform; the first method utilizes SMART (Switching Mechanism At 5' end of RNA Transcript) technology, while the second involves replacing the 5' cap structure with RNA oligomers via ligation. The third and fourth methods are modifications of SMART, and involve enriching mRNA m ...[more]

PMID: 25003736

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Project description:BackgroundPost-transcriptional gene regulation is one of the critical layers of overall gene expression programs and microRNAs (miRNAs) play an indispensable role in this process by guiding cleavage on the messenger RNA targets. The transcriptome-wide cleavages on the target transcripts can be identified by analyzing the degradome or PARE or GMUCT libraries. However, high-throughput sequencing of PARE or degradome libraries using Illumina platform, a widely used platform, is not so straightforward. Moreover, the currently used degradome or PARE methods utilize MmeI restriction site in the 5' RNA adapter and the resulting fragments are only 20-nt long, which often poses difficulty in distinguishing between the members of the same target gene family or distinguishing miRNA biogenesis intermediates from the primary miRNA transcripts belonging to the same miRNA family. Consequently, developing a method which can generate longer fragments from the PARE or degradome libraries which can also be sequenced easily using Illumina platform is ideal.ResultsIn this protocol, 3' end of the 5'RNA adaptor of TruSeq small RNA library is modified by introducing EcoP15I recognition site. Correspondingly, the double-strand DNA (dsDNA) adaptor sequence is also modified to suit with the ends generated by the restriction enzyme EcoP15I. These modifications allow amplification of the degradome library by primer pairs used for small RNA library preparation, thus amenable for sequencing using Illumina platform, like small RNA library.ConclusionsDegradome library generated using this improved protocol can be sequenced easily using Illumina platform, and the resulting tag length is ~ 27-nt, which is longer than the MmeI generated fragment (20-nt) that can facilitate better accuracy in validating target transcripts belonging to the same gene family or distinguishing miRNA biogenesis intermediates of the same miRNA family. Furthermore, this improved method allows pooling and sequencing degradome libraries and small RNA libraries simultaneously using Illumina platform.

Dataset Information

Four methods of preparing mRNA 5' end libraries using the Illumina sequencing platform.

Publications

Four methods of preparing mRNA 5' end libraries using the Illumina sequencing platform.

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