Psylliodes chrysocephala adult transcriptome annotation

1.1 Library preparation <br> The total RNA from pre-aestivation (5-day-old), aestivation (30-day-old), and post-aestivation (55-day-old) female beetles were extracted using ZYMO Quick-RNA Tissue/Insect Kit (ZYMO Research, Irvine, CA, USA) and cleaned using TURBO DNA-free™ kit (Thermo Fisher Scientific, Langenselbold, Germany) according to the manufacturer’s instructions. We opted to sample only the females to eliminate sex-related variations. RNA quantity was determined using a Nanodrop ND-1000 UV/Vis spectrophotometer (Thermo Fisher Scientific). The integrity of the RNA samples was determined using the Agilent 2100 Bioanalyzer and an RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA, USA). RIN values ≥ 7.0 were considered appropriate for mRNA library preparation. In total, 10 libraries (4, 3, and 3 libraries respectively per pre-aestivation, aestivation, and post-aestivation stages) were prepared using NEBNext® Poly(A) mRNA Magnetic Isolation Module kit (NEB E7490, New England Biolabs) according to the manufacturer’s instructions. The qualities of the libraries were checked via RNA fragment analysis conducted on the Agilent 2100 Bioanalyzer using the Agilent DNF-935 Reagent Kit (Agilent Technologies). The libraries were pooled based on their concentration, and an overall concentration of 3.4 ng/µL was obtained. The sequencing service was provided by BGI Genomics Tech Solutions Co. Ltd (Hong Kong) on a DNBSEQ-T7 platform. <br> 1.2 <em>De novo</em> assembly and functional annotation <br> Erroneous k-mers from paired read ends were removed using r-Corrector (v1.0.5) the with default options (Song &amp; Florea, 2015), and the unfixable reads were discarded using the “FilterUncorrectabledPEfastq.py” function in Transcriptome Assembly Tools (Song &amp; Florea, 2015). The adaptor sequences from the reads were removed, and the reads having a quality score above 30 were retained using TrimGalore! (v0.6.7). The cleaned reads (n = 3 per three adult phases) were <em>de novo</em> assembled using Trinity with default options. The completeness of the transcriptome was quantified using BUSCO (v5.4.2) via a comparison against the endopterygota dataset (BUSCO.v4 datasets). The transcriptome (including isoforms) was annotated using Trinotate (v3.2.2), which combines the outputs of NCBI BLAST+ (v2.13.0; nucleotide and predicted protein BLAST), TransDecoder (v5.5.0; coding region prediction), signal (v4.0; signal peptide prediction), TmHMM (v2.0; transmembrane domain prediction), and HMMER (v3.3.2; homology search) packages into an SQLite annotation database. The latest uniport_sprot (04/2022) and Pfam-A (11/2015) databases were downloaded using Trinotate, and the default E-value thresholds were used during the searches with BLAST+ and HMMER, respectively. The obtained annotation database was used to extract gene ontology (GO) terms associated with individual genes using the “extract_GO_assignments_from_Trinotate_xls.pl” whereas the signals and TmHMM outputs were manually extracted using Excel spreadsheets. The longest protein-coding regions in the super transcript data predicted by TransDecoder were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation via GhostKoala v2.2 (https://www.kegg.jp/ghostkoala/)