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Small RNA-seq: The RNA 5'-end adapter ligation problem and how to circumvent it.

J Biol Methods. 2019; 
LamaLodoe,CoboJose,BuenaventuraDiego,RyanK
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PCR Cloning and Subcloning … 2.5% agarose gel, and the bands visualized using ethidium bromide, excised and extracted using QuickClean II Gel Extraction Kit (Genscript, L00418). The extracted libraries were ligated into a linearized T-vector [20]. Five clones were picked and sequenced (Macrogen USA) … Get A Quote

摘要

The preparation of small RNA cDNA sequencing libraries depends on the unbiased ligation of adapters to the RNA ends. Small RNA with 5' recessed ends are poor substrates for enzymatic adapter ligation, but this 5' adapter ligation problem can go undetected if the library preparation steps are not monitored. Here we illustrate the severity of the 5' RNA end ligation problem using several pre-miRNA-like hairpins that allow us to expand the definition of the problem to include 5' ends close to a hairpin stem, whether recessed or in a short extension. The ribosome profiling method can avoid a difficult 5' adapter ligation, but the enzyme typically used to circularize the cDNA has been reported to be biased, ... More

关键词

CircLigase,RNA ligase bias,TS2126,coligo,small RNA
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