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

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 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.