Gills
RNA was isolated from frozen tissues using TRIzol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturers guidelines.  RNA integrity was checked using Bioanalyzer (Agilent, https://www.agilent.com/).  RNA samples with RIN > 9 were used for Iso-Seq library preparation.  The Clontech SMARTer PCR cDNA Synthesis Kit was used for first strand cDNA synthesis according to Pacific Biosciences instructions. Following PCR Cycle Optimization, a large-scale PCR was performed to generate double-stranded cDNA for SMRTbell library construction.  AMPure PB Bead Purification of large-Scale PCR Products was performed exonuclease was used to remove failed ligation products.  AMPure PB beads were used to purify SMRTbell Templates twice.   The sequencing libraries were prepared by annealing a sequencing primer and binding a polymerase to SMRTbell templates.  In total, 19 SMRT cells were sequenced.
Gills_m54120_190507_221302.subreads.bam