For sequencing the embryonic transcriptome, we extracted total RNA of mixed stage embryos of the wt (until egg stage 12 [staging according to Donoughe and Extavour, 2016], combined into one sample, known as Gb1) and egg stages 1–3 (combined into one sample, known as Gb2) of the pXLBGact Histone2B:eGFP strain (Nakamura et al., 2010). An ovary was dissected out of an adult female, and total RNA was extracted as described below.

For total RNA extraction, 50–100 mg of embryos or ovaries were homogenised in 1 ml of TRIZOL. After centrifugation for 15 min at 4°C and 12,000 g, the supernatant was transferred to a fresh tube and 200 µl chloroform/1 ml of TRIZOL was added. Vortexing for 15 s and centrifugation for 15 min at 4°C and 12,000 g led to a phase separation. The total RNA of the upper aqueous phase was cleaned using the Zymoclean Quick RNA MicroPrep Kit according to the manufacturer's protocol. Library preparation (including poly A selection) and stranded sequencing was carried out at the Cologne Center for Genomics (HiSeq2000 for the embryonic and HiSeq4000 for the ovarian transcriptome).

We also downloaded previous sequencing reads from transcriptomic studies of G. bimaculatus (Fisher et al., 2018) from the sequence read archive (SRA). The previously sequenced reads and those from our sample were subjected to adaptor and quality threshold trimming using Trimmomatic 0.33 (Bolger et al., 2014), the appropriate adaptor sequences and the following quality cutoffs: LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:30. FastQC (Andrews, 2010) was used to assess read quality both before and after trimming. Raw reads from our sequencing are available from the NCBI SRA under accession PRJNA492804.

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