RNA sequencing library construction using single-stranded ligation of a DNA adapter to 3' ends of cDNAs often produces primer-adapter byproducts, which compete with cDNA-adapter ligation products during library amplification and, therefore, reduces the number of informative sequencing reads. We find that Escherichia coli Exo I digestion efficiently and selectively removes surplus reverse transcription primer and thereby reduces the primer-adapter product contamination in 3' cDNA ligation-based sequencing libraries, including small RNA libraries, which are typically similar in size to the primer-adapter products. We further demonstrate that Exo I treatment does not lead to trimming of the cDNA 3' end when duplexed with the RNA template. Exo I digestion is easy to perform and implement in other protocols and could facilitate a more widespread use of 3' cDNA ligation for sequencing-based applications.

中文翻译：

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Exo I对引物的过度降解改善了基于3'cDNA连接的测序文库的制备。

使用DNA衔接子与cDNA的3'端单链连接的RNA测序文库构建通常会产生引物-适配器副产物，其在文库扩增过程中与cDNA-适配器的连接产物竞争，因此减少了信息性测序读数的数量。我们发现大肠杆菌Exo I消化可有效，选择性地去除多余的逆转录引物，从而减少基于3'cDNA连接的测序文库中的引物-接头产物污染，包括小RNA库，该文库的大小通常与引物-接头产物相似。我们进一步证明，Exo I处理与RNA模板双工时不会导致cDNA 3'末端的修剪。Exo I消化易于在其他方案中执行和实施，并且可以促进3'cDNA连接在基于测序的应用中的更广泛使用。