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.