Transcriptome Analysis of Insect Embryogenesis

We collected a series of samples in three replicates to isolate total RNA to cover developmental periods before and after the primary sex-determination event in embryos. Samples include ~200 embryos collected at each stage (i.e., 0–1 h, 2–4 h, 4–8 h, and 8–12 h post-oviposition), along with 15 sex-mixed pupae, 15 male adults, and 15 female adults. RNA concentrations and purity were determined initially by Nanodrop 2000 spectrophotometry (Thermo Fisher Scientific, USA). Samples of OD260:280 > 2.0 were submitted to BGI Tech for further quality control using the Agilent 2100 Bioanalyzer. A RIN (RNA integrity number) value >8.0 was met to process library construction. Libraries were constructed and sequenced with 150 bp paired ends on BGISEQ-500 (MGI, Shenzhen, China), yielding a total of 718,007,728 reads (each sample >20 million reads). All RNA-seq reads were input to Trinity assembler v2.11.0^{36 (link)} to run a de novo assembly with default parameters to create the transcriptome. Based on the assembled transcriptome, transcript levels were quantified and normalized to TMM (trimmed mean of M-values) using the abundance_estimates_to_matrix.pl scripts available in the trinity toolkit^{36 (link)}. The resulting data were submitted to the NCBI SRA database (PRJNA834573).