tailfindr: Alignment-free poly(A) length measurement for Oxford Nanopore RNA and DNA sequencing

Maximilian Krause; Adnan M. Niazi; Kornel Labun; Yamila N. Torres Cleuren; Florian S. Müller; Eivind Valen

To generate RNA with known poly(A) tail lengths, we used eGFP as a carrier RNA as it fulfils basic criteria for successful ONT RNA sequencing (especially minimal length requirement). The coding sequence of eGFP was amplified from pCS2+-eGFP vector using High Fidelity Phusion MasterMix (ThermoFisher, #F-531L). The primers for the PCR included the SP6 promoter sequence and a barcode in the forward primer, as well as the a homopolymer T stretch in the reverse Primer (see Table 1 ). After gel-purification of the desired PCR product, a second PCR was performed with a reverse Primer that introduces a Bfo1 restriction site before the homopolymer T stretch (polyA Bfo1 rev, together with SP6 Bfo1 fw, Table 1 ). After gel-purification and Phenol-chloroform extraction, the resulting PCR products were used for Nanopore DNA Ligation Sequencing (see below). For preparation of RNA spike-ins, the PCR products were digested with FastDigest Bfo1 (ThermoFisher, #FD2184) for 2 hours and purified by Phenol-chloroform extraction.