Capturing 5' and 3' native ends of mRNAs concurrently with Akron sequencing

Advances in RNA-sequencing methods have uncovered many aspects of RNA metabolism but are limited to surveying either the 3' or 5' terminus of RNAs, thus missing mechanistic aspects that could be revealed if both ends were captured. We developed Akron sequencing (Akron-seq), a method that captures in parallel the native 5' ends of uncapped, polyadenylated mRNAs and 3' ends of capped mRNAs from the same input RNA. Thus, Akron-seq uniquely enables assessment of full-length and truncated mRNAs at single-nucleotide resolution. Akron-seq involves RNA isolation, depletion of ribosomal and abundant small capped RNAs, and selection of capped and polyadenylated mRNAs. The endogenous ends of mRNAs are marked by adaptor ligation, followed by fragmentation, cDNA generation, PCR amplification, and deep sequencing. The step-by-step protocol we describe here is optimized for cultured human cells but can be adapted to primary cells and tissues. Akron-seq can be completed within 6 d, and sequencing and analysis can be completed within 6 d.