GSM4910654: HiC.dom-kd.st5.rep2; Drosophila melanogaster; Hi-C
50 µl ice-cold HiC lysis buffer (10 mM Tris-HCl [pH 8], 10 mM NaCl, 0.2% Igepal, 1x Protease Inhibitor Cocktail (Roche, 11836170001)) were added and embryos were crushed with a pestle. Then, nuclei were pelleted by centrifugation (3000g, 5min at 4°C). Next, the pellet was incubated in 50 µl 0.5% SDS for 10 min at room temperature. The reaction was quenched by adding 145 µl of water and 25 µl of 10% Triton X100. For chromatin digestion, 0.7 µl of DpnII (NEB R0543T) and 25 µl of DpnII buffer were added. The reaction was incubated for 90 min at 37°C at 600 rpm and a second instance of 0.7 µl of DpnII was added followed by a second incubation of 90 min. Nuclei were then pelleted and resuspended in 135 µl of 1x CutSmart buffer (NEB B7204S). To biotinylate the free chromatin ends, 0.15mM (final) of dNTP-mix (NEB), 0.05mM (final) of Biotin-14-dATP (Jena Bioscience, NU-835-BIO14-S) and 10 U of Klenow (NEB M0210 L) was added to the reaction and incubated for 60 min at 25 °C. The chromatin free ends were then ligated by adding ligation buffer (NEB B0202S) (1x final concentration), TritonX-100 (0.8% final concentration), 120 µg BSA and 2000 U ligase (NEB M0202S). This reaction was incubated for 2hrs, after which another 2000 U of ligase were added and incubated for another 2hrs. Nuclei were then pelleted and resuspended in 200 µl elution buffer (10 mM Tris-HCl [pH 8], 1 mM EDTA, 1% SDS). Proteins were digested upon addition of 400 µg Proteinase K for 30 min at 55 °C. The chromatin was reverse crosslinked in the presence of 0.365 M NaCl at 68 °C overnight. DNA was purified using the ChIP DNA Clean&Concentrator Kit (Zymo Research D5205) eluted in 50 µl and biotin was removed from non-ligated fragments by adding NEB buffer 2 (NEB B7002S) (1x final), 12 µg of BSA, dATP and dGTP (0.025 final each), 3 U T4 DNA polymerase (NEB M0203S) in a final volume of 120 µl. The reaction was stopped by adding a final concentration of 13 mM EDTA. Samples were sheared with Covaris E220 (Covaris microTUBE snap cap (520045) with intensifier (50014), duty factor 10%, peak incident power 140, cycle/burst 200, 120 sec) to generate fragments of 200-300bp. Ligated and biotinylated fragments were enriched through biotin pulldown using Dynabeads MyOne Streptavidin C1 (Invitrogen 65001). Afterwards, 2x binding buffer (10 mM Tris-HCl [pH 8], 1 mM EDTA, 2 M NaCl) (1x final concentration) was added to each sample. Subsequently, beads (equilibrated with TWB (5 mM Tris-HCl [pH 8], 0.5 mM EDTA, 1 M NaCl, 0.05% Tween) resuspended in 1x binding buffer were added to each sample. The beads were washed twice for 2 min at 55 °C with TWB, twice with EB-buffer (Qiagen), resuspended in 50 µl of EB-buffer and directly used for library preparation Libraries were prepared using the NEB Ultra II DNA Library Prep Kit for Illumina (E7645S and E6440) following the manufacturer's instructions except that the adapter ligation occurred on the biotin beads. The fragments were released from the beads before the PCR reaction (by heating to 98 °C for 10 min) once non-ligated adapters had been removed.