Excess primer degradation by Exo I improves the preparation of 3′ cDNA ligation-based sequencing libraries

Christel Hougård Enroth, Annaleigh Ohrt Fehler, Line Dahl Poulsen, Jeppe Vinther
2019 BioTechniques  
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
more » ... equencing 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.
doi:10.2144/btn-2018-0178 pmid:31208218 fatcat:ogrbcgr6tnerho2f2cjehxj3ca