miRNA Biogenesis by Ulf Andersson Vang Ørom

Author:Ulf Andersson Vang Ørom
Language: eng
Format: epub, pdf
Publisher: Springer New York, New York, NY

Here, we describe a protocol for cDNA library preparation from pre-miRNA (and mature miRNAs) for subsequent high-throughput sequencing using the Illumina platform. This protocol does not require the use of radioactivity, minimizes ligation biases by using linkers with random nucleotide sequences at the RNA-ligation interface and diminishes the generation of “empty” adapter dimers, which can compromise library amplification yield [17, 26]. For the purpose of candidate pre-miRNA analysis, we also present a targeted pre-miRNA cDNA library preparation protocol, based on previously described protocols [24, 25]. Finally, we provide details on the bioinformatics parameters routinely used for the analysis of Drosophila miRNA and pre-miRNA tailing events, by means of the publicly available computational pipeline “Tailor” [27].

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