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Targeted Methods to Improve Small RNA Profiles Generated by Deep Sequencing

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Regulatory RNAs

Abstract

Several recent reviews expertly address the relative merits of different approaches to preparation and analysis of deep-sequenced small RNA libraries. Here, we focus on an array of protocols and tools with the intention of assisting researchers in improving short RNA profiles constructed with second-generation sequencing. This includes methods and commentaries on the preparation of sequencing-caliber immunoprecipitation RNA libraries, techniques for targeting different populations of RNAs with distinct 5′- and 3′-ends, reduction of adapter dimers in libraries, and dealing with the underappreciated problem of genomic cross-mapping of similar miRNA sequences.

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Abbreviations

AGO:

Argonaute

BAP:

Bacterial alkaline phosphatase

bp:

Base pairs

CAGE:

Cap analysis gene expression

cDNA:

Complementary DNA

CLIP-seq:

Cross-linked immunoprecipitation and deep sequencing of RNAs

HITS-CLIP:

High-throughput sequencing of RNAs isolated by cross-linked immunoprecipitation

IP:

Immunoprecipitation

LNA:

Locked nucleic acid

miRNA:

microRNA

mRNA:

Messenger RNA

nt:

Nucleotides

PAR-CLIP:

Photoactivatable-ribonucleoside-enhanced cross-linking and immunoprecipitation

PASR:

Promoter-associated small RNA

piRNA:

Piwi-interacting RNA

qPCR:

Quantitative real-time PCR

RISC:

RNA-induced silence complex

siRNA:

Small interfering RNA

T4 PNK:

T4 polynucleotide kinase

TAP:

Tobacco acid pyrophosphatase

TASR:

Termini-associated small RNA

TEX:

TerminatorTM 5′-phosphate-dependent exonuclease

UTR:

Untranslated region

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Acknowledgments

We thank Takahiro Nishibu, Ryo Ukekawa, Taku Funakoshi, and Tsutomu Kurokawa of Wako Pure Chemical Industries, Ltd. and Carsten Daub of the RIKEN Omics Science Center (OSC) for their collaboration. This work was supported by a research grant for the RIKEN OSC from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) to YH and a grant for the Innovative Cell Biology by Innovative Technology (Cell Innovation Program) from the MEXT to YH.

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  1. Omics Science Center (OSC), RIKEN Yokohama Institute, Yokohama, Kanagawa, Japan

    Yoshinari Ando, A. Maxwell Burroughs, Mitsuoki Kawano, Michiel Jan Laurens de Hoon & Yoshihide Hayashizaki

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  1. Yoshinari Ando
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  2. A. Maxwell Burroughs
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  3. Mitsuoki Kawano
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  4. Michiel Jan Laurens de Hoon
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  5. Yoshihide Hayashizaki
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Correspondence to A. Maxwell Burroughs .

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  1. , New York State Department of Health, Wadsworth Center, New Scotland Avenue 120, New York, 12208, New York, USA

    Bibekanand Mallick

  2. A.J.C. Bose Centenary Building, P-1/12, CIT Scheme - VII M, Bose Institute, Kolkata, 700054, India

    Zhumur Ghosh

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Ando, Y., Burroughs, A.M., Kawano, M., de Hoon, M.J.L., Hayashizaki, Y. (2012). Targeted Methods to Improve Small RNA Profiles Generated by Deep Sequencing. In: Mallick, B., Ghosh, Z. (eds) Regulatory RNAs. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-22517-8_10

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