Deoxyribonuclease 1

Total RNA was isolated using TRIzol, treated with deoxyribonuclease I (New England Biolabs, M0303S), and reverse transcribed using SuperScript III and random primers (Invitrogen). Resulting cDNA was analyzed by quantitative PCR using Green SuperMix for iQ (VVR, 01414-144) on a 7500 Fast Real-Time PCR system (Thermo Fisher Scientific) or on a CFX ConnectTM Real-Time PCR Detection System (Bio-Rad).

Total RNA from four minifish individuals were extracted separately using the TRIzol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. The RNA was treated with deoxyribonuclease I (New England Biolabs, Ipswich, MA, USA) before cDNA synthesis. Fifty percent of the total RNA extracted from each fish (~3 to 8 μg) was used for cDNA synthesis in reactions containing no more than 0.5 μg of total RNA. cDNA synthesis was performed using the SMARTScribe Reverse Transcriptase (Clontech, Mountain View, CA, USA) with an oligo-dT primer (5′-AAGCAGTGGTATCAACGCAGAGTTTTTTTTTTTTTTTTTTTTTTTTVN) and SMARTer_Oligo_UMI primer (5′-AAGCAGUGGTAUCAACGCAGAGUNNNNUNNNNUNNNNUCTT[rGrGrGrGrG]) according to the SMARTer RACE 5’RACE protocol (Clontech, Mountain View, CA, USA). The SMARTer_Oligo_UMI is a hybrid primer with riboguanosines representing the last five bases and the remainder representing deoxyribonucleotides, including the U (deoxyuracil); the Ns represent the bar code. The cDNA synthesized was treated with uracil-DNA glycosylase before all reactions from the same individual were combined together. The combined cDNA was purified using the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany), eluted with 70 μl of diethyl pyrocarbonate (DEPC)–treated water, and vacuum-dried.

Dysf-3xHA (UAS-dysf-3xHA, tubGAL80^ts) was overexpressed by slbo-GAL4 in follicle cells. Female flies were kept at 25°C for 4 days and then shifted to 31°C for 1 day before dissection. We dissected 400 pairs of ovaries from female flies overexpressing UAS-dysf-3xHA in Drosophila S2 cell medium (Thermo Fisher Scientific) and then lysed them in 200 μl of radioimmunoprecipitation assay buffer (50 mM tris-Cl, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, and 1× EDTA-free protease inhibitor). The ovary lysate was kept on ice and sonicated (ChromTech UP-500) for 10 cycles at 30% amplitude for 5 s on/10 s off. After sonication, lysates were centrifuged at 15,000g at 4°C for 20 min. The supernatant was applied for pull-down assay. Tissue was harvested from 24B-GAL4>UAS-dysf-3xHA, tubGAL80^ts larvae maintained at 25°C for 4 days and then shifted to 31°C for 1 day before dissection and extraction of Dysf-3xHA. Tissue was harvested from stat92e-stat92e::gfp larvae kept at 25°C for 5 days before dissection and extraction of Stat::Flag. Salivary glands and imaginal discs from 200 larvae were dissected in S2 cell medium and lysed as described above for ovary extract preparation. All lysates were incubated with deoxyribonuclease I (6 U/ml; NEB) at 4°C for 30 min before undergoing pull-down assay.