title: IsoSeq of Actias luna: fourth instar silk gland
design: Silk glands were carefully extracted from a single individual at each representative life stage (instars 1-5  L1-5) by decapitating caterpillars live over ice while submerged in Qiagen RNAprotect Tissue Reagent (Qiagen, Cat #76104). Glands were immediately flash frozen in liquid nitrogen in 1.8 mL microcentrifuge tubes and stored at -80C. RNA extractions, library preparation, sequencing and initial subread processing were performed at the University of Floridas Interdisciplinary Center for Biotechnology Research (RRID:SCR_019152). RNA was isolated using a Qiagen RNeasy micro extraction kit (Qiagen, Cat #74004) following manufacturer protocol and assessed for concentration and quality using a Qubit fluorometer and the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc). SMRT Bell IsoSeq libraries were prepared for PacBio SEQUEL IIe according to manufacturer protocol (PacBio, Cat #PN 101-763-800) with few modifications. RNA preps were cleaned and concentrated using the ZYMO Research RNA Clean and Concentrator kit (ZYMO Research, Cat #R1015). Total RNA from each larval instar silk gland was diluted to approximately 100 ng, after which full-length cDNA was synthesized with amplification as described in the protocol for Low Input RNA:cDNA Synthesis and Amplification (New England Biolabs, Cat #E6421). Further sample prep was performed with PacBio SMRTbell Express Template Prep Kit 2.0 (PacBio, PN 100-938-900) and Barcoded Overhang Adapter Kits A&B (PacBio, Cat #PN 100-628-400 and #PN 100-628-500), with 1 g of amplified cDNA per sample as input. The final library contained one barcoded cDNA sample per larval instar, for a total of five samples. The barcoded library was sequenced using the PacBio Sequel IIe platform. Consensus sequences for subreads were generated using ccs. All further subread processing was performed with the IsoSeq 3 pipeline (v4.0.0, https://github.com/PacificBiosciences/IsoSeq). Lima v2.7.1 was used to filter reads and remove primers and barcodes for each sample separately.
library_name: ACLU0128