GSM4173929: HiC WT stage 5 repl1; Drosophila melanogaster; Hi-C
Embryos were defrosted in ice cold HiC lysis buffer (10 mM Tris-HCl [pH 8], 10 mM NaCl, 0.2% Igepal,   1x Protease Inhibitor Cocktail (Roche, 11836170001)) on ice and immediately crashed with a pestle for 30 sec. The crashing of the embryos was checked under a microscope. The nuclei were pelleted for 5 min at 3000 g (rcf) and 4 °C. After discarding the supernatant the pellet was incubated in 50 µl 0.5% SDS for 10 min at RT. The reaction was quenched by adding 145 µl of water and 25 µl of 10% Triton X100.   To digest the chromatin 25 µl of DpnII buffer were added and 0.7 µl of DpnII (NEB R0543T). The reaction was incubated for 90 min at 37 °C in a thermomixer at 600 rpm and a second instance of 0.7 µl of DpnII was added followed by a second incubation of 90 min.    The nuclei were pelleted for 5 min at 1000 xg (rcf) and RT and resuspended in 135 µl of 1x CutSmart buffer (NEB B7204S). To biotinylate the free chromatin ends, dNTP-mix (NEB) (final 0.15 mM), Biotin-14-dATP (Jena Bioscience, NU-835-BIO14-S) (final 0.05 mM) and 10 U of Klenow (NEB M0210 L) were added to the reaction (final volume 150 µl) and incubated for 60 min at 25 °C. Next the free ends of the chromatin were ligated in a total volume of 1200 µl by adding ligation buffer (NEB B0202S) (1x final), 10% Triton (0.8% final), 120 µg BSA, 2000 U ligase (NEB M0202S). After two hours a second instance of 2000 U ligase was added and incubated for another 2 hours. Following the incubation the nuclei were pelleted at 1000 g for 5 min at RT, the supernatant was discarded and the nuclei 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.   The next day DNA was purified using the ChIP DNA Clean&Concentrator Kit (Zymo Research D5205) eluted in 50 µl and biotin was removed from unligated 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 EDTA to a final concentration of 13 mM.  The DNA was transferred to Covaris microTUBE snap cap (520045) and sheared using the Covaris E220 (with intensifier (50014) and the following parameters duty factor 10%, peak incident power 140, cycle/burst 200, 120 sec. The DNA fragments were in the range of 200-300 bp.  Ligated and biotinylated fragments were enriched through biotin pulldown using Dynabeads MyOne Streptavidin C1 (Invitrogen 65001). The samples were filled up to a total of 400 µl with 2x binding buffer (10 mM Tris-HCl [pH 8], 1 mM EDTA, 2 M NaCl) (1x final) and 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. The beads were washed twice for 2 min at 55 °C with TWB and twice with EB-buffer (Qiagen). Last the beads were resuspended in 50 µl of EB-buffer and directly used for library preparation using the NEB Ultra II DNA Library Prep Kit for Illumina (E7645S) following the manufacturer's instructions except that the adapter ligation happened on the biotin beads. The fragments were released from the beads before the PCR reaction (by heating to 98 °C for 10 min) after unligated adapters had been removed. To amplify the libraries 12-13 cycles of PCR were performed. We used NEB Ultra II DNA Library Prep Kit for Illumina (E7645S) following the manufacturer's instructions except that the adapter ligation happened on the biotin beads.
GEO
GSM4173929
GEO Accession
GSM4173929