Transcriptome Analysis of Insect Tissues

Salivary tissues and midguts were preserved in TRIzol (Thermo-Fisher, Waltham, MA). Salivary glands were excised as described by Cicero and Brown [67 (link)] in pools of 150 per replicate in TRIzol LS (Thermo-Fisher, Waltham, MA). Samples were kept at −80°C (bioreps 1–3, CLas (-/+) were kept one year, while replicate 4, both CLas (-/+), was kept for two years) prior to RNA extraction. Total RNA was extracted for both midguts and salivary glands following the standard TRIzol (Thermo-Fisher, Waltham, MA) RNA extraction protocol [68 (link)] including light syringe disruption prior to adding ethanol, and DNase treatment to purify total RNA. Total RNA quality was tested using an RNA gel prior to library preparation. Details of midgut sample handling can be found in Kruse et al. [16 (link)].
Illumina (San Diego, CA) libraries for all samples were made by Polar Genomics LLC (Ithaca, NY) following the protocol of Zhong et al. [69 (link)] and included poly-A tailed mRNA enrichment. Libraries were shipped on dry ice to GENEWIZ (Azenta Life Sciences, Plainfield, NJ), where they were pooled for Illumina (San Diego, CA) paired-end (PE) 150 bp sequencing. Bacteriome, head, and salivary gland samples were sequenced separately from the previously published midgut samples [16 (link)]. Raw data have been uploaded to NCBI and are accessible to reviewers via BioProject accession No. PRJNA385527.