GSM9290594: wtOSC_HiC; Drosophila melanogaster; Hi-C
Two OSC aliquots (5 × 10⁶ cells each; 1 × 10⁷ total) were processed in parallel. Cells were resuspended in 1 mL ice-cold lysis buffer, incubated for 30 min at 4 °C with rotation, and pelleted (2,500 g, 5 min, 4 °C). Pelleted Nuclei were resuspended in 1 mL lysis buffer, pelleted again, and finally resuspended in 400 µL 1.2× NEB buffer 3.1, minimizing additional wash steps to reduce clumping. Nuclei were digested overnight at 37 °C with rotation (800 rpm) using 30 U DpnII (NEB) in a final volume of 80 µL. After digestion, pellets were resuspended in 52 µL fill-in mix containing 2.2 µL biotin-14-dATP (1 mM, Jena Bioscience), 1.1 µL dNTP mix (2 mM dCTP, dGTP, dTTP each), 11 µL Klenow fragment, 5.72 µL NEB buffer 2, and nuclease-free water to volume, and incubated for 1.5 h at 37 °C with rotation. Filled-in nuclei were resuspended in a 250 µL ligation mix (27.5 µL Thermo T4 DNA ligase buffer, 2.2 µL 10% Triton X-100, 2.25 µL BSA [10 mg/mL], 232.5 µL H₂O, and 11 µL T4 DNA ligase). Samples were incubated at RT for 5 h with rotation. Aliquots (10 µL) were collected at digestion and ligation steps as process controls. Post-ligation, reactions were diluted to 500 µL with PBS, supplemented with NaCl (final concentration TBD) and 10 µL proteinase K (20 mg/mL), and incubated overnight at 65 °C. DNA was purified by two rounds of phenol–chloroform extraction, followed by a chloroform wash, and ethanol precipitation in the presence of glycogen. Pellets were resuspended in 150 µL H₂O (controls in 10 µL), treated with RNase A (2 µL; 10 U/µL, 30 min, 37 °C), and quantified using Qubit. 130 microliters of DNA were transferred to Covaris microTUBEs and sheared on a Covaris E220 (<10 °C, intensifier inserted, 105 W peak power, 5% duty factor, 200 cycles/burst, 55 s). DNA was rinsed from Covaris tubes with 80 µL EB and subjected to two-step Ampure XP bead size selection (0.44× and 0.18×). DNA was eluted in 100 µL H₂O. Biotinylated DNA was captured with Dynabeads MyOne Streptavidin C1, pre-washed in Tween wash buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20). Beads were resuspended in 100 µL 2× binding buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2 M NaCl), mixed with DNA, and incubated for 1.5 h at RT with rotation. Beads were washed twice in 500 µL Tween wash buffer and twice in H₂O, and resuspended in 50 µL H₂O. Bead-bound DNA was subjected to end repair with NEBNext Ultra II End Prep (7 µL End Prep buffer, 3 µL enzyme mix; 30 min at 20 °C then 30 min at 65 °C), adaptor ligation (2.5 µL NEB Illumina adaptor, 1 µL ligation enhancer, 30 µL ligation master mix; 15 min at 20 °C), and USER enzyme treatment (3 µL; 15 min, 37 °C). Beads were washed four times with 500 µL Tween wash buffer. DNA was eluted in 20 µL pre-warmed elution buffer (10 mM EDTA, 95% formamide, 65 °C), ethanol-precipitated with glycogen and NaCl, and resuspended in 15 µL H₂O. PCR amplification was performed with KAPA HiFi polymerase (25 µL master mix, 2 µL EvaGreen, 2.5 µL 10 µM barcoded primer, 1 µL 25 µM universal forward primer, 4.5 µL water). Cycling proceeded for 5 cycles, stopping when real-time fluorescence monitoring reached 3,600 RFU. PCR products were diluted, purified using Zymo DNA Clean & Concentrator-5, and eluted in 15 µL H₂O. Final libraries were quantified with Qubit, quality-checked on a Fragment Analyzer, and sequenced (PE75, illumina NextSeq 550).