Easy blue system

RNA extraction and complementary DNA (cDNA) synthesis of fly samples were performed as previously described (Chung et al., 2017 (link), Lee et al., 2023 (link), Lee et al., 2022 (link)). Total RNAs were extracted from adult fly heads using Easy-Blue system (iNtRON Biotechnology). cDNAs were synthesized from 3 µg of total RNAs using GoScript Reverse Transcription (Promega, A2791) according to the manufacturer’s standard protocol. Quantitative PCR experiments were performed using the synthesized cDNAs with QuantiSpeed SYBR Green kit (PhilKorea, QS105-10) and CFX96 TouchTM Real-Time PCR Detection System (Bio-Rad). Gene-specific primers were listed in Supplementary Figure S5a (Tran et al., 2015 (link), Xu et al., 2013 (link)). rp49 was used as a housekeeping gene to normalize the expression levels of target genes. mRNA fold change of each gene was calculated using the comparative Ct method. Gene-specific primers were designed with Primer-BLAST (ncbi.nlm.nih.gov/tools/primer-blast) and Drosophila RNAi Screening Center FlyPrimerBank (flyrnai.org/flyprimerbank). Some primer sequences were obtained from published studies.

RT-PCR analysis was performed as previously described (Chung et al., 2017 (link)). Total RNAs were extracted from adult fly heads using Easy-Blue system (iNtRON Biotechnology). cDNAs were synthesized from 3 µg of total RNAs using GoScript Reverse Transcription (A2791; Promega) according to the manufacturer’s standard protocol. For RT-PCR analysis, each target gene was amplified with the corresponding primer set (Table S2 and Table S3) using GoTaq G2 Master Mixes (M7823; Promega) in a C1000 Thermal Cycler, C1000 Touch Thermal Cycler, or T100 Thermal Cycler system (Bio-Rad).