title: iso-seq dusky lory
design: Total RNA was isolated using the RNeasy kit (Qiagen, Valencia, CA) following the manufacturers protocol. RNA degradation and contamination were monitored on an Agilent RNA ScreenTape and RNA concentration was measured using Quant-iT RiboGreen RNA Assay Kit in Victor Nivo (PerkinElmer, Waltham, MA, USA). The integrity of RNA was assessed using the RNA ScreenTape of the Agilent 2200 TapeStation System (Agilent Technologies, CA, USA). A total of 300ng RNA was used for full-length transcript sequencing with Pacbio Sequel IIe system (Pacific Biosciences, CA, USA) according to the manufacturers instructions. The Iso-Seq library was prepared according to the Isoform Sequencing (Iso-Seq) protocol using the NEBNext Single Cell/Low Input cDNA Synthesis & Amplification Module. A total of 10 uL of library was prepared using SMRTbell prep kit 3.0. SMRTbell templates were annealed using Sequel II binding kit 3.1. The Sequel II sequencing kit 2.0 and SMRT cells 8M Tray were used for sequencing. SMRT cells (Pacific Biosciences) using 24 hr movies were captured for each SMRT cell using the PacBio Sequel IIe (Pacific Biosciences) sequencing platform by Macrogen (Seoul, Korea). The subsequent steps are based on the PacBio Sample Net-Shared Protocol, which is available at https://www.pacb.com/.