ChIP-seq protocol for Drosophila L3 EDs

ChIP–seq on L3 EDs were performed as described previously^{41 (link)}, with minor modifications, and 400 EDs were used per replicate. If necessary, several dissection and/or collection batches were frozen in liquid nitrogen and stored at −80 °C to collect sufficient material. Chromatin was sonicated using a Bioruptor Pico (Diagenode) for 10 min (30 s on, 30 s off). PH antibodies^{67 (link)} were diluted 1:100 for immunoprecipitation. After decrosslinking, DNA was purified using MicroChIP DiaPure columns from Diagenode. DNA libraries for sequencing were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina. Sequencing (paired-end sequencing 150 bp, roughly 4 Gb per sample) was performed by Novogene (https://en.novogene.com/). All experiments were performed in biological duplicates.