The concomitant cloning of RNA degradation products is a major concern in standard small RNA-sequencing practices. This not only complicates the characterization of bona fide sRNAs but also hampers cross-batch experimental replicability and sometimes even results in library construction failure. Given that all types of plant canonical small RNAs possess the 3′ end 2′-O-methylation modification, a new small RNA sequencing (sRNA-seq) method, designated as PBOX-sRNA-seq, has been developed specifically to capture this modification. PBOX-sRNA-seq, as its name implies, relies on the sequential treatment of RNA samples with phenylboronic acid-polyacrylamide gel electrophoresis (PBA-PAGE) and sodium periodate (NaIO4) oxidation, before sRNA library construction and sequencing. PBOX-sRNA-seq outperformed separate treatments (i.e. PBA-PAGE only or NaIO4 only) in terms of the depletion of unmethylated RNA species and capture 2′-O-modified sRNAs with extra-high purity. Using PBOX-sRNA-seq, we discovered that nascent miRNA-5p/-3p duplexes may undergo mono-cytidylation/uridylation before 2′-O-methylation. We also identified two highly conserved types of 5′-tRNA fragments (tRF) bearing HEN1-independent 2′-O modification (mainly the 13-nt tRF-5aAla and the 26-nt tRF-5bGly). We believe that PBOX-sRNA-seq is powerful for both qualitative and quantitative analyses of sRNAs in plants and piRNAs in animals.

中文翻译：

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PBOX-sRNA-seq 揭示了 miRNA 修饰的新特征并鉴定了带有 2'-O 修饰的选定 5'-tRNA 片段


RNA 降解产物的伴随克隆是标准小 RNA 测序实践中的一个主要问题。这不仅使真实 sRNA 的表征变得复杂，而且阻碍了跨批次实验的可重复性，有时甚至导致文库构建失败。鉴于所有类型的植物经典小 RNA 都具有 3' 端 2'-O-甲基化修饰，因此专门开发了一种新的小 RNA 测序 (sRNA-seq) 方法，称为 PBOX-sRNA-seq，用于捕获这种修饰。 PBOX-sRNA-seq，顾名思义，依赖于在 sRNA 文库构建和测序之前，使用苯基硼酸-聚丙烯酰胺凝胶电泳 (PBA-PAGE) 和高碘酸钠 (NaIO4) 氧化对 RNA 样品进行顺序处理。 PBOX-sRNA-seq 在未甲基化 RNA 种类的耗尽和以超高纯度捕获 2'-O 修饰的 sRNA 方面优于单独处理（即仅 PBA-PAGE 或仅 NaIO4）。使用 PBOX-sRNA-seq，我们发现新生的 miRNA-5p/-3p 双链体可能在 2'-O-甲基化之前经历单胞苷化/尿苷化。我们还鉴定了两种高度保守类型的 5'-tRNA 片段 (tRF)，其带有不依赖于 HEN1 的 2'-O 修饰（主要是 13-nt tRF-5aAla 和 26-nt tRF-5bGly）。我们相信 PBOX-sRNA-seq 对于植物中的 sRNA 和动物中的 piRNA 的定性和定量分析都很强大。