2.5 Hi-C library preparation
The Hi-C library preparation was constructed following a previous protocol (Zhuang et al. 2019), Briefly, A single 3rd-instar larva was washed, and after removal of the dissected guts, the remaining tissues were fixed with 1% formaldehyde. Subsequently, glycine was added to quench the cross-linking reaction, and tissues were finally ground to powder and suspended in nuclei isolation buffer to obtain a nuclei suspension (Belton et al. 2012). The nuclei mixture was dissolved, the chromatin was digested with restriction enzyme (Dpn II), and end-labeled by incubating with Klenow enzyme and biotin-14-dCTP generating blunt-end-repaired DNA strands (Lieberman-Aiden et al.2009), and ligated by T4 DNA polymerase. The extracted DNA was mechanically sheared to 200–300 bp sizes. DNA fragments of 150–300 bp were blunt-end repaired and A-tailed, followed by purification through biotin–streptavidin-mediated pulldown (Burton et al. 2013). PCR amplification was performed after adapters were ligated to the Hi-C products. The PCR products were purified with AMPure XP beads, and the Hi-C libraries were quantified by quantitative PCR reaction (Lieberman-Aiden et al. 2009). The Hi- C library was constructed by the NEBNext Ultra II DNA library Prep Kit and then sequenced (150 bp paired-end reads) on the Illumina HiSeq Xten.