Total RNA was extracted from the spiked-in cell mixture using hot acid phenol. The quality of extracted RNA was assessed by gel electrophoresis (1% TBE gel), and concentrations were determined by Qubit RNA HS kit (Invitrogen). DNase treatment of the RNA samples was done on-column using RNase-Free DNase Set (QIAGEN) followed by rRNA-depletion using S. cerevisiae riboPOOLs probes (siTOOLs Biotech). Sequencing libraries were prepared using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina (NEB #E7760) coupled with AMPure XP bead purification (Beckman Coulter) with 18-22 ng of rRNA-depleted RNA as an input. Library quality and fragment distribution were assessed on an Agilent fragment analyzer.