direct cDNA-seq of E. coli DSM 30083 ATCC 11775
Escherichia coli strain ATCC 11775 was cultured overnight at 37C in 25 mL of Luria broth (LB) and following centrifugation the cell pellet was resuspended in 250 L, to which 750 L of TRIzol reagent (Life Technologies, Carlsbad, CA, USA) was added. Following incubation for 5 minutes at room temperature, 200 L of chloroform were added. Phases were mixed by inverting the tube 15 times and then incubated for 10 min. Following centrifugation at 12,000 x g for 5 min at 4C, 400 L of the aqueous phase was removed and the RNA it contained was cleaned using the Direct Zol kit (Zymo Research).  Poly(A)-tailing reaction using E. coli EPAP (NEB# M0276) was designed to perform in a 26 l reaction (reaction size can be scaled up as needed). A total of 500 ng total E. coli RNA was used to prepare dcDNA-seq library. The dcDNA library was produced using the SQK-DCS108 kit (ONT, Oxford, UK) which includes an RT step. Direct sequencing was performed on a single R9.5/FLO-MIN107 flow cell.