2.5 RNA-seq and transcriptome data processing
A. flavipes were obtained by overnight live-trapping in Samford
Valley, QLD, Australia (27° 22′ S, 152° 52′ E). Animals were euthanised
by CO2 and tissues frozen at -80 °C. Eight specimens
collected in 2017 were sequenced by BGI Hong Kong. The RNA of the
remainder [seven males and six females collected in 2018] was
extracted by BGI Hong Kong and sequenced by BGI Qingdao. Only the
samples collected during the 2018 breeding season in Samford Valley were
used for differential gene expression analysis (the samples collected in
2017 were used for gene annotation only). Raw data was filtered using
Flexbar v3.4.0 (Dodt, Roehr, Ahmed, & Dieterich, 2012; Roehr,
Dieterich, & Reinert, 2017) with default settings (removes reads with
any uncalled bases). BGISEQ-500 adapters were obtained from a
comparative study of Illumina and BGI sequencing platforms (Mak et al.,
2017). Any residual ribosomal RNA reads (the majority removed by poly(A)
selection prior to sequencing library generation) were removed from the
reads using SortMeRNA v2.1b (Kopylova, Noe, & Touzet, 2012) against the
SILVA v119 ribosomal database (Quast et al., 2013). Tissue
transcriptomes were de novo assembled using Trinity v2.8.4 (Grabherr et
al., 2011; Haas et al., 2013; Henschel et al., 2012) with default
parameters. The A. flavipes transcriptome assemblies were
assessed using BUSCO (Seppey et al., 2019). Details of transcriptome
sequencing data used in study are listed in Tables S2-S4 (also
see Supplemental Methods).