Small interspersed elements (SINEs) are transcribed by RNA polymerase III (Pol III) to produce RNAs typically 100–500 nucleotides in length. Although their RNA abundance can be evaluated by Northern blotting and primer extension, the nature (sequence, exact length, and genomic origin) of these RNAs cannot be revealed by these methods. Moreover, mRNA sequencing (mRNA-seq) is not able to distinguish bona fide SINE RNAs or SINE sequences present in longer transcripts. To elucidate the abundance, source loci, and sequence nature of SINE RNAs, we established a deep sequencing method, designated as melRNA-seq (medium-length RNA-seq), which can determine whole-length RNA sequences. Total RNA samples were treated with 5′ pyrophosphohydrolase (RppH), which allowed ligation of an RNA adaptor to the 5′ end of intact SINE RNAs. Similarly, another adaptor was ligated to the 3′ end, followed by reverse transcription, PCR amplification, size selection, and single-end deep sequencing. The analysis of two biological replicates of RNAs from mouse spermatogonia showed high reproducibility of SINE expression data both at family and locus levels. This new method can be used for quantification and detailed sequence analysis of medium-length non-coding RNAs, such as rRNA, snRNA, tRNAs, and SINE RNAs. Further, its dynamic range is much wider than Northern blotting and primer extension.

中文翻译：

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melRNA-seq 用于 SINE RNA 和其他中等长度非编码 RNA 的表达分析

小的散在元件 (SINE) 由 RNA 聚合酶 III (Pol III) 转录，以产生长度通常为 100-500 个核苷酸的 RNA。尽管可以通过 Northern 印迹和引物延伸来评估它们的 RNA 丰度，但这些方法无法揭示这些 RNA 的性质（序列、确切长度和​​基因组起源）。此外，mRNA 测序 (mRNA-seq) 无法区分存在于较长转录物中的真正的 SINE RNA 或 SINE 序列。为了阐明 SINE RNA 的丰度、来源位点和序列性质，我们建立了一种深度测序方法，称为 melRNA-seq（中长 RNA-seq），可以确定全长 RNA 序列。总 RNA 样品用 5' 焦磷酸水解酶 (RppH) 处理，这允许将 RNA 接头连接到完整 SINE RNA 的 5' 末端。相似地，将另一个接头连接到 3' 端，然后进行逆转录、PCR 扩增、大小选择和单端深度测序。对来自小鼠精原细胞的 RNA 的两个生物学重复的分析表明，SINE 表达数据在家族和基因座水平上具有很高的重现性。这种新方法可用于中等长度非编码 RNA（如 rRNA、snRNA、tRNA 和 SINE RNA）的定量和详细序列分析。此外，它的动态范围比 Northern 印迹和引物延伸要宽得多。这种新方法可用于中等长度非编码 RNA（如 rRNA、snRNA、tRNA 和 SINE RNA）的定量和详细序列分析。此外，它的动态范围比 Northern 印迹和引物延伸要宽得多。这种新方法可用于中等长度非编码 RNA（如 rRNA、snRNA、tRNA 和 SINE RNA）的定量和详细序列分析。此外，它的动态范围比 Northern 印迹和引物延伸要宽得多。