GSM9150978: WD, Hi-C, control, rep2; Drosophila melanogaster; Hi-C
200 wing discs for each replicate were quickly dissected (<1 hour) from wandering larvae at room temperature (RT) in Schneider's insect medium before being directly processed. Wing discs were washed twice in PBS and resuspended in A1 Buffer (60 mM KCl, 15 mM NaCl, 4 mM MgCl2, 15 mM HEPES, 0.5% Triton-X-100, 0.5 mM DTT, 1x Protease Inhibitor Cocktail (Sigma-Aldrich), H2O). Then, formaldehyde was added to reach a 1.8% final concentration and transferred on a rotating wheel for 10 minutes at RT. Formaldehyde was quenched with 1.25 M glycine and samples were washed twice with ice-cold A1 Buffer. Supernatant was discarded and fixed wing discs were air dried and then snapped frozen in liquid nitrogen to process all samples in parallel. For all samples, wing discs were thawed on ice and resuspended in ice-cold A1 Buffer. The samples were homogenized on ice using a tight Tenbroeck, centrifuged and resuspended in 0.5% SDS for 10 min at 62°C to permeabilize nuclei. SDS was quenched by adding Triton-X-100 and incubated for 15 min at 37°C with rotation. Next, samples were resuspended in lysis buffer for a 30 min incubation on ice and nuclei were subjected to Mbo I treatment O/N at 37°C in NEB2.1 buffer. The next day, restriction sites were end-repaired and biotinylated using Klenow [New England BioLabs (NEB), catalog no. M0210] and biotin-14-dATP (Life Technologies, catalog no. 19524-016) before being re-ligated using T4 DNA ligase (NEB, catalog no. M0202) O/N at 16 ºC. Next day, samples were treated with RNAse A for 15 min at 37ºC and then incubated with proteinase K and reverse crosslinked for 6 h at 65°C. Subsequently, DNA was purified using AMPure XP beads (Beckman CoulterTM, catalog no. A63880) and then sheared to obtain fragments of an average size of 300–400 bp using the Bioruptor Pico (Diagenode; 1µg of DNA in 100µl; 6 cycles; 20'' ON, 60'' OFF). For library preparation, biotinylated DNA was pulled down by adding an equal volume of Dynabeads MyOne Streptavidin T1 beads (Life Technologies, catalog no. 65602). Biotin was removed from unligated ends, and pulled-down DNA fragments were end-repaired and A-tailed with NEBNext A-tailing module (E6053L). Illumina adaptors were ligated, and libraries were amplified by eight cycles of PCR using NEBNext High-Fidelity 2X PCR Master Mix (catalog no. M0541S). DNA was size selected using AMPure XP beads (Beckman CoulterTM, catalog no. A63880) at a ratio of beads to library of 0.9:1 to isolate 300- to 800-bp fragments. Lastly, the quality of the libraries was visualized with the Agilent Bioanalyzer High Sensitivity DNA Assay (Agilent Technologies, Savage, DE) and checked by low sequencing depth on a NextSeq 500 prior to higher sequencing depth on NovaSeq6000 platform at the Centre for Genomic Regulation (CRG) sequencing facility in Barcelona, Spain. A minimum of 500 million paired-end 150-bp-long reads were obtained per sample.