Efficient microRNA capture and bar-coding via enzymatic oligonucleotide adenylation

Vigneault, François; Sismour, A. Michael; Church, George M.

doi:10.1038/nmeth.1244

Cited by 48 publications

(44 citation statements)

References 13 publications

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“…Linkers were pre-adenylated as previously described with some modifications (Vigneault et al 2008). Eight hundred pmols of RNA linker was incubated for 5 h at 37°C in a reaction containing 200 units of T4 RNA ligase I [New England Bio labs (NEB)], 13 RNA ligase I buffer [50mM Tris-HCl pH 7.8 / 10mM MgCl 2 / 1mM ATP / 10mM DTT], 4 units of Ribolock RNase A inhibitor (Fermentas), and 25% DMSO.…”

Section: Methodsmentioning

confidence: 99%

Deep sequencing of microRNA precursors reveals extensive 3′ end modification

Newman¹,

Mani²,

Hammond³

2011

RNA

107

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MicroRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally regulate gene expression. An emerging mechanism to control miRNA production is the addition of an oligo-uridine tail to the 39 end of the precursor miRNA. This has been demonstrated for the Let-7 family of miRNAs in embryonic cells. Additionally, nontemplated nucleotides have been found on mature miRNA species, though in most cases it is not known if nucleotide addition occurs at the precursor step or at the mature miRNA. To examine the diversity of nucleotide addition we have developed a high-throughput sequencing method specific for miRNA precursors. Here we report that nontemplated addition is a widespread phenomenon occurring in many miRNA families. As previously reported, Let-7 family members are oligo-uridylated in embryonic cells in a Lin28-dependent manner. However, we find that the fraction of uridylated precursors increases with differentiation, independent of Lin28, and is highest in adult mouse tissues, exceeding 30% of all sequence reads for some Let-7 family members. A similar fraction of sequence reads are modified for many other miRNA families. Mono-uridylation is most common, with cytidine and adenosine modification less frequent but occurring above the expected error rate for Illumina sequencing. Nucleotide addition in cell lines is associated with 39 end degradation, in contrast to adult tissues, where modification occurs predominantly on full-length precursors. This work provides an unprecedented view of the complexity of 39 modification and trimming of miRNA precursors.

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Section: Methodsmentioning

confidence: 99%

Deep sequencing of microRNA precursors reveals extensive 3′ end modification

Newman¹,

Mani²,

Hammond³

2011

RNA

107

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“…The increasing read depth for sequencing prompted the development of cost-saving multiplexing of samples by barcoding approaches (Parameswaran et al 2007;Vigneault et al 2008;Xu et al 2009;Buermans et al 2010;Schulte et al 2010;Farazi et al 2011). It is preferable to place the barcoding step as early as possible in the RNA processing protocol to minimize parallel handling of samples rather than at the end stage of cDNA library preparation as in current commercial kits (Illumina TruSeq Small RNA Sample Prep kit).…”

Section: Guide To Reagents and Critical Experimental Stepsmentioning

confidence: 99%

RNA-ligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries

Hafner¹,

Renwick²,

Brown³

et al. 2011

RNA

307

367

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Sequencing of small RNA cDNA libraries is an important tool for the discovery of new RNAs and the analysis of their mutational status as well as expression changes across samples. It requires multiple enzyme-catalyzed steps, including sequential oligonucleotide adapter ligations to the 39 and 59 ends of the small RNAs, reverse transcription (RT), and PCR. We assessed biases in representation of miRNAs relative to their input concentration, using a pool of 770 synthetic miRNAs and 45 calibrator oligoribonucleotides, and tested the influence of Rnl1 and two variants of Rnl2, Rnl2(1-249) and Rnl2(1-249)K227Q, for 39-adapter ligation. The use of the Rnl2 variants for adapter ligations yielded substantially fewer side products compared with Rnl1; however, the benefits of using Rnl2 remained largely obscured by additional biases in the 59-adapter ligation step; RT and PCR steps did not have a significant impact on read frequencies. Intramolecular secondary structures of miRNA and/or miRNA/39-adapter products contributed to these biases, which were highly reproducible under defined experimental conditions. We used the synthetic miRNA cocktail to derive correction factors for approximation of the absolute levels of individual miRNAs in biological samples. Finally, we evaluated the influence of 59-terminal 5-nt barcode extensions for a set of 20 barcoded 39 adapters and observed similar biases in miRNA read distribution, thereby enabling cost-saving multiplex analysis for large-scale miRNA profiling.

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“…Concatemerization of the preadenylated linker is prevented by blocking with 3 ′ end by addition of either a dideoxy nucleotide or an amine group. The amine blocker is equally effective and less expensive (Vigneault et al 2008). One and one-half micrograms of total RNA in a total volume of 10 µL was denatured for 10 min at 65°C and then cooled on ice for 2 min.…”

Section: Rna 3 ′ End Ligationmentioning

confidence: 99%

EnD-Seq and AppEnD: sequencing 3′ ends to identify nontemplated tails and degradation intermediates

Welch

Slevin

Tatomer³

et al. 2015

RNA

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Existing methods for detecting RNA intermediates resulting from exonuclease degradation are low-throughput and laborious. In addition, mapping the 3 ′ ends of RNA molecules to the genome after high-throughput sequencing is challenging, particularly if the 3 ′ ends contain post-transcriptional modifications. To address these problems, we developed EnD-Seq, a high-throughput sequencing protocol that preserves the 3 ′ end of RNA molecules, and AppEnD, a computational method for analyzing highthroughput sequencing data. Together these allow determination of the 3 ′ ends of RNA molecules, including nontemplated additions. Applying EnD-Seq and AppEnD to histone mRNAs revealed that a significant fraction of cytoplasmic histone mRNAs end in one or two uridines, which have replaced the 1-2 nt at the 3 ′ end of mature histone mRNA maintaining the length of the histone transcripts. Histone mRNAs in fly embryos and ovaries show the same pattern, but with different tail nucleotide compositions. We increase the sensitivity of EnD-Seq by using cDNA priming to specifically enrich low-abundance tails of known sequence composition allowing identification of degradation intermediates. In addition, we show the broad applicability of our computational approach by using AppEnD to gain insight into 3 ′ additions from diverse types of sequencing data, including data from small capped RNA sequencing and some alternative polyadenylation protocols.

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Efficient microRNA capture and bar-coding via enzymatic oligonucleotide adenylation

Cited by 48 publications

References 13 publications

Deep sequencing of microRNA precursors reveals extensive 3′ end modification

Deep sequencing of microRNA precursors reveals extensive 3′ end modification

RNA-ligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries

EnD-Seq and AppEnD: sequencing 3′ ends to identify nontemplated tails and degradation intermediates

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