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 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.