A diversity of RNA molecule 5' ends are generated during transcriptional and post-transcriptional processes. Different RNA ends can confer or represent different functional activities and thus the identification of RNA end usage dynamics contributes to the functional characterization of RNA molecules. Here we present a method that enables the accurate identification of RNA 5' ends from samples with low amounts of total RNAs, and thus allow characterization of RNA regulatory mechanisms in specific cell-types. Overall design: NanoPARE, or RNA 5' end enrichment, libraries were constructed for three biological replicates of ten conditions/genotypes. mRNA-Seq libraries were generated were constructed for three biological replicates of two conditions/genotypes.