Gel-free library preparation for next-generation RNA sequencing and small RNA quantification

Next-generation RNA sequencing (RNA-seq) is hampered by “primer dimer” (PD) artifacts and its quantitative performance reduced by polymerase fall-off (PF) at RNA modifications and secondary structures. Here we improved RNA-seq efficiency by incorporating (i) a post-reverse-transcription (RT) digestion of excess primers with Escherichia coli exonuclease I for PD mitigation, thus obviating gel purification during RNA-seq library preparation, and (ii) a high-processivity reverse transcriptase to increase full-length reads. A full factorial experimental design was applied to absolute quantification RNA sequencing (AQRNA-seq), the most accurate NGS-based method for quantifying small RNAs, using cDNA libraries constructed from E. coli small RNAs (>85% tRNA) followed by sequencing, data processing, and data analysis. The novel PF and PD mitigation approaches increased AQRNA-seq sensitivity >10-fold by minimizing PF and maximizing target RNA reads. By increasing sensitivity and obviating gel electrophoresis for removing PD, AQRNA-seq and other NGS-based RNA-seq methods can now be automated to increase throughput and reduce RNA sample size. Overall design: cDNA libraries for Escherichia coli strain BW25113 small RNAs were constructed following four variations of the library preparation protocols with three biological replicates. Constructed libraries were subjected to quality assessment using Fragment Analyzer and qPCR, followed by paired-end sequencing (PE75) on a Illumina MiSeq platform.