Improved full‐length cDNA production based on RNA tagging by T4 DNA ligase

Published online January 2, 2004 Nucleic Acids Research, 2004, Vol. 32, No. 1 e6 DOI: 10.1093/nar/gng158 Improved full-length cDNA production based on RNA tagging by T4 DNA ligase Christian Clepet*, Isabelle Le Clainche and Michel Caboche Unite de Recherches en GeÂnomique VeÂgeÂtale, INRA/CNRS, 2 Rue Gaston-CreÂmieux, F-91057 Evry Cedex, France Received September 16, 2003; Revised and Accepted October 30, 2003 ABSTRACT INTRODUCTION The study of full-length cDNAs remains an indispensable approach for structural and functional genome annotations (Castelli,V., Aury,J.-M., Jaillon,O., Wincker,P., Clepet,C., Menard,M., Cruaud,C., SchaÈchter,V., Temple,G., Caboche,M., Weissenbach,J. and Salanoubat,M., submitted). The wide range of methods described in patents and the scienti®c literature shows how critical but dif®cult this research area is. The central problem every full-length cDNA method tries to resolve is grafting a known sequence at the cap site, so as to be able to prime second-strand polymerisation of the cDNA. In some methods, cap-dependent tagging is used as a way of selecting for full-length cDNAs; in other protocols the tag is added on cDNAs previously enriched for molecules extending to the 5¢cap (1,2). A number of enzymatic or chemical taggings have been described, either on single-strand cDNA (see for example 3,4), double-strand cDNA (see for example 5,6), de-capped mRNA (see for example 7±9) or straight on the mRNA cap (10,11). In the strategy of Sekine and Kato (8) and of Maruyama and Sugano (9), RNAs are dephosphorylated by alkaline phosphatase, decapped by tobacco acid pyrophosphatase and ligated to an oligonucleotide by T4 RNA ligase. This method is one of the most speci®c and accessible for full-length cDNA production, however its ef®ciency is somewhat limited at the RNA ligation step. Tavitgian et al. (12) and Shibata et al. (13) used T4 DNA ligase for oligonucleotide joining on single-strand cDNA. The procedure is based on using partially degenerate MATERIALS AND METHODS Preparation of the cap adapter The cap adapter was prepared by mixing 40 mM of both oligonucleotides P1 and P2, in a buffer containing 10 mM *To whom correspondence should be addressed. Tel: +33 160874512; Fax: +33 160874510; Email: Second-strand cDNA priming is a central problem for full-length characterization of transcripts. A new strategy using bacteriophage T4 DNA ligase and partially degenerate adapters is proposed for grafting a sequence tag to the end of polyribonucleotides. Based on this RNA tagging system and previously described protocols, a new method for full-length cDNA production has been implemented. Validation of the method is shown in Arabidopsis thaliana by the construction of a full-length cDNA library and the analysis of 154 clones and by 5¢-RACE±PCR run on a documented experimental system. adapters to create a local double-stranded structure at the junction between the oligonucleotide 5¢-phosphate and the cDNA 3¢-OH. This tagging does not discriminate against incomplete reverse transcription products and needs, as in Shibata et al. (13), to be used in conjunction with other enrichment methods for obtaining full-length cDNAs. T4 DNA ligase (once known as polynucleotide ligase) can catalyse DNA-templated joining of RNA fragments (14,15). Based on this property, a new full-length cDNA strategy has been designed. After alkaline phosphatase inactivation of uncapped nucleic acids and cap removal by tobacco acid pyrophosphatase, an oligonucleotide is speci®cally grafted to the cap site of mRNAs. In contrast to previous methods (8,9), RNA tagging is performed by using T4 DNA ligase and adapters generating local double-stranded structure at the junction with the mRNA 5¢-end. The ligated mRNAs can then be used for RACE±PCR or library construction. Gateway attB sites have been included in the 5¢ RNA adapter and the 3¢ reverse transcription oligo(dT) primer, enabling cDNA library construction by recombinational cloning (16). As a proof of concept, 5¢ RACE±PCR and full-length cDNA library analyses have been performed in Arabidopsis thaliana. The overall sensitivity of the strategy is shown in a convergent way by both experimental approaches. In particular, the 5¢ RACE±PCR revealed a new upstream transcription start site (TSS) for ats1A. The constructed full-length cDNA library has been evaluated by comparison with the genomic annotations and the cDNA catalogue available for A.thaliana. In particular, amongst the 154 clones analysed, a 5¢-end located upstream of the AGI (17) annotated coding sequence (CDS) was found for 130 cDNAs and, when compared to EMBL sequences, 12 inserts showed a sequence gain towards the promoter. Besides the production of full-length cDNAs, this new RNA tagging strategy could be used in any application where an oligo needs to be grafted on one or both sides of RNA fragments of unknown sequence. In particular, it could be useful for amplifying RNA targets isolated by ribonucleoprotein immunoprecipitation experiments (18). e6 Nucleic Acids Research, 2004, Vol. 32, No. 1 NaCl and 10 mM Tris±HCl (pH 7.5), heating at 70°C in a beaker of water and left to hybridise by cooling down to room temperature. The adapter was dispensed into small aliquots and kept at ±80°C. RNA Total RNAs were extracted by the Trizol method (Invitrogen) from 4-week-old A.thaliana aerial vegetative tissues, according to the manufacturer's recommendations. Cap-site tagging of mRNAs Decapping. The RNA was then digested for 1 h at 37°C with 2 U tobacco acid pyrophosphatase (Epicentre) in 20 ml of its accompanying buffer supplemented by 40 U RNasin. The reaction volume was made up to 100 ml with H2O, phenolchloroform extracted and ethanol precipitated as above. The RNA pellet was washed with 75% ethanol and air dried for 5 min. Oligonucleotide ligation. The decapped RNA was resuspended in 6.5 ml of H2O, heated at 65°C for 5 min, equilibrated at 25°C and mixed in a 10ml ®nal volume with 50 mM Tris± HCl (pH 7.5), 10 mM MgCl2, 10 mM dithiothreitol (DTT), 1 mM ATP, 25 mg/ml of bovine serum albumin (BSA), 5% polyethylene glycol 8000, 20 U Rnasin, 4 mM double-stranded cap adapter and 1000 U highly concentrated T4 DNA ligase (New England Biolabs). The reaction was incubated for 3 h at 25°C. The reaction volume was made up to 100 ml with H2O, phenol-chloroform extracted and precipitated with 300 mM sodium acetate, 20 mg of glycogen and 2 vol of ethanol, overnight at ±20°C. The RNA pellet was washed with 75% ethanol and air dried for 5 min. Reverse transcription The RNA pellet was resuspended in 8 ml of H2O, mixed with P3 primer (50 pmol) and dNTPs (10 nmol each) in a 12 ml ®nal volume and heated at 65°C for 5 min. The solution was equilibrated at 48°C and completed with 40 U RNasin, 10 mM DTT, 50 mM Tris±HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2 and 200 U M-MLV Superscript III (Invitrogen), in 20 ml ®nal volume. Reverse transcription was performed for 50 min at 48°C and stopped by heatin (...truncated)