direct cDNA-seq of yeast S288C
Yeast RNA was isolated from S. cerevisiae strain S288C grown overnight at 30C in 15 mL medium containing 10 g/L yeast extract, 20 g/L peptone, and 20 g/L glucose. RNA was extracted using the ZymoBIOMICS Quick-RNA Fungal/Bacterial kit (Zymo Research, Irvine, CA, USA). A total of 600 ng total yeast RNA was used to prepare librariy. The dcDNA library was produced using the SQK-DCS108 kit (ONT, Oxford, UK) which includes an RT step but no amplification step. RNA was then converted to double stand DNA, after which ligation of the adaptor attached the motor protein. The library was loaded onto a flow cell for a 48 hour sequencing run lasting. Direct sequencing was performed on a single R9.5/FLO-MIN107 flow cell.