PacBio Iso-Seq of Diorhabda sublineata: heads
RNA extraction was performed from three female heads using Trizol and the Zymo Direct-zol 96 MagBead RNA kit. These heads, cDNA was synthesized from the RNA using the NEBNext Single Cell/Low Input cDNA Synthesis and Amplification kit using the PacBio Iso-Seq Express Template Switching Oligo. The Iso-Seq library was prepared from cDNA with the PacBio Iso-Seq Express Library Preparation Using SMRTbell Express Template Prep Kit 2.0. Sequencing was performed on a Sequel II System using Binding Kit v2.0, Sequencing kit v2.0, and SMRT Cell 8M. To target HiFi reads, four libraries were barcoded individually and pooled for sequencing using a 30 hour movie time on one SMRTcell. Raw subreads were converted to HiFi data by processing with CCS to call a single high quality consensus sequence for each molecule, using a 99.5% consensus accuracy cutoff.