2006 Kirino and Mourelatos 2007 Pak and Fire 2007 Batista et al. However, many small RNA species contain modifications at the ends ( Chen 2005 Ruby et al. In the ligation-based methods, the 5′ ligation needs a 5′ monophosphate (p) and the 3′ ligation needs a 3′OH on target RNA. mRNA can be fragmented and then used to make cDNA with 5′ and 3′ linkers added using various methods including ligation and reverse transcription (RT) small RNA is usually first ligated with 5′ and 3′ linkers and then converted to cDNA. To clone RNA, RNA is converted to cDNA with 5′ and 3′ linkers added for cDNA amplification and sequencing. The DNA library construction strategy is straightforward, basically ligating fragmented-and-end-fixed target DNA with partially double-stranded DNA linkers, as used by commercial kits ( Illumina 2012 Kircher et al. In most sequencing platforms, a DNA or cDNA library is made by ligating fragmented DNA/RNA with platform-specific linkers, which are then used to design primers for amplifying and reading sequences/barcodes ( Goodwin et al. If a laboratory wants to prepare its own barcoded PCR primers, it has to prepare three sets. Since the linkers for DNA, mRNA, and RNA libraries are different, the corresponding PCR primers for obtaining the final amplicons are also different. Most commercial kits only provide just enough primers for a specified number of library constructions, allowing no mistakes or errors during cloning. Lack of compatibility among these expensive kits and transparency for the protocol details often lead to confusion and waste. Usually DNA, mRNA, and small RNA sequencing libraries are constructed with distinct linkers and/or chemistry, forcing customers to purchase different kits ( Reuter et al. Although this strategy significantly decreases the sequencing cost, it cannot solve the problem that the overall library construction cost using commercial kits could easily surpass the sequencing cost. Since a single sequencing run is usually sufficient for analyzing multiple samples, individual samples are usually cloned with specific barcodes, pooled, and sequenced as a single library for cost-sharing, and then debarcoded to obtain sample-specific sequences ( Stiller et al. Therefore, any technical advance in this technique will have a broad impact. As the cost has significantly decreased over the past decade, it is becoming a standard tool for gene analyses in biomedical fields. High-throughput sequencing has become a revolutionary technique for analyzing DNA/RNA. Moreover, the all-liquid-based reaction can be performed in an automated manner. Not only is our method more convenient for cloning modified RNA than available methods, but it is also more sensitive, versatile, and cost-effective. Moreover, this method can also clone mRNA, simplifying the need to prepare two cloning systems for small RNA and mRNA the barcoded PCR primers are also compatible with non-cDNA cloning applications, including the preparation of genomic libraries. The 7-h cloning process only needs ∼1 h of labor. Here we present a new strategy to clone 5′ modified or unmodified small RNA in an all-liquid-based reaction carried out in a single PCR tube with as little as 20 ng total RNA. Unlike mRNA, small RNA often contains modifications including 5′ cap or triphosphate and 2′- O-methyl, requiring additional processing steps before linker additions during cloning processes due to low expression levels, it is difficult to clone small RNA with a small amount of total RNA. This method usually needs a cDNA/DNA library ligated with specific 5′ and 3′ linkers. High-throughput sequencing has become a standard tool for analyzing RNA and DNA.
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