Testis dissection and total RNA extraction

The transcriptome analysis was performed using the testes of N. vitripennis pupae at two developmental stages: the white nymph stage (during which the primary spermatocytes are the only germ cells present) and at the red-eyed white nymph stage (during which the secondary spermatocytes are the only germ cells present) [23]. To represent each stage, the testes of 50–60 males (i.e., 100–120 testes) were dissected in RNA-free water, immediately snap frozen in liquid nitrogen, and stored at -80 °C until RNA extraction could take place. Three pools per condition were used. RNA was extracted from the samples using a NucleoSpin® RNA XS Kit (Macherey–Nagel gmbH & Co. KG, Germany) and an adapted version of the manufacturer’s instructions. The RNA extractions were then eluted in 11 µl of RNAse-free water and stored at -80 °C until the transcriptome analysis took place. Total RNA concentration was quantified using a Qubit® 3.0 fluorometer (Thermo Fisher Scientific, Waltham, MA) and a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA integrity was verified using an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA).

Samples were sent to GenoScreen (Lille, France) for sequencing. Sample quality was verified beforehand using a Quant-iT™ RiboGreen® RNA Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), a DNF-471 RNA Analysis Kit (15 nt, standard sensitivity; Advanced Analytical Technologies, Ankeny, USA), and a fragment analyzer.

To generate RNA-seq libraries, the Illumina TruSeq RNA Sample Preparation Kit (Illumina, San Diego, California) was used in accordance with the manufacturer’s instructions. Briefly, mRNA was purified from 200 ng of the total RNA from each sample, enriched using polyT marble, and fragmented. Then, mRNA was converted into double-stranded cDNA. Sequencing barcodes were ligated to the cDNA fragments, and the resulting fragments were amplified using PCR. Libraries were validated using the DNF-474 High-Sensitivity NGS Fragment Kit and a fragment analyzer to confirm that the libraries had the expected fragment size of ~ 350 bp. Finally, SYBR™ Green I dye was used to ensure adequate library concentrations.

Sequencing libraries were multiplexed such that each multiplex contained one library from each of the two developmental stages and were then sequenced using a HiSeq 2500 System (rapid run mode, 2 × 100 bp; Illumina, San Diego, California). For each sample, we obtained sequences ranging from 1,040 to 3,136 Mbp.