RNA-seq Analysis of Drosophila Embryos

Embryos were collected at 25°C (5-16 h after egg laying) and dechorionated in 2% sodium hypochlorite solution for 4 min. After subsequent washing with embryo wash (0.8% NaCl and 0.05% Triton X-100) and H₂O, embryos were stored at −80°C. One embryo collection for three biological replicates per genotype was obtained. RNA-extraction was carried out according to the manufacturer's protocol (Promega ReliaPrepTM RNA Tissue Miniprep System, REF-Z6111). Total RNA was measured using NanoDrop OneC (Thermo Scientific) for its concentration and RNA integrity was checked using gel electrophoresis. Then 9-15 µg of total RNA/biological replicate was shipped to Novogene for sequencing. Prior to making the library, samples were reassessed for quality with the Agilent 2100 Bioanalyzer system. Sequencing was performed on an Illumina platform and paired-end reads were produced. Over 40 million reads/genotype were generated and mapped to the genome at a rate of over 96%. Drosophila melanogaster (ensemble bdgp6_gca_000001215_4 genome assembly) was used. HISAT2 algorithm for alignment and DESeq2 R package (Anders and Huber, 2010 (link)) for differential gene expression was used. Subsequent analyses were performed with GraphPad Prism 9. Fold change ≥1.5 and ≤−1.5 (log2FC≥0.59 and ≤−0.59) for up- and downregulated genes, respectively, and P_adj≤0.05 were used for statistical significance.