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. University of Pennsylvania researchers have 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 the researchers 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.
Overview of Akron-seq workflow
After total RNA extraction from HeLa cells and bead-size selection to remove small RNAs (≤200 nt; Steps 1–21), we split the sample in two tubes (Step 22) for the generation of Akron3 (Step 22A) and Akron5 (Step 22B) libraries. First, we enrich for capped mRNAs by Terminator exonuclease treatment after depletion of rRNAs and snRNAs (Akron3). Then we select polyadenylated mRNAs (Akron5) by binding on oligo(dT)25 beads followed by capture of native ends by adaptor ligation, fragmentation, second adaptor ligation, and generation of Illumina-compatible libraries by reverse transcription and PCR amplification (Steps 23–44, see Procedure for specific details). B, a biotin-TEG group; CAP, the 5ʹ-cap modification; N, the 3ʹ ends of non-polyadenylated RNAs; other, the uncapped RNA ends; P, a phosphate group; Phos., phosphorylation; X, a blocking group.