GSM2977368: S2 control_Hi-C_rep1; Drosophila melanogaster; Hi-C
Hi-C analysis was performed as described previously (Belton et al. 2012), with the minor modifications described below. A total of 3-5 million cells were pelleted and resuspended in 10 ml of 1× PBS (10 mM phosphate buffer, 2.7 mM KCl 137 mM NaCI, pH7.4) and fixed with 1% formaldehyde (Sigma) for 10 min at room temperature. The reaction was stopped by adding 2 M glycine to a final concentration of 125 mM. The cell suspension was incubated on ice for 5 min, and the cells were pelleted (10 min at 1000 x g in a pre-chilled (4oC) centrifuge). The supernatant was removed, and the cell pellet was washed with 500 μl of 1× PBS, frozen and, if necessary, stored in a liquid nitrogen. After thawing, the cells were lysed for 15 min in 1.5 ml of ice-cold lysis buffer (50 mM Tris-HCl (pH8.0), 150 mM NaCl, 0.5% (v/v) NP-40, 1% (v/v) Triton-X100, 1× Protease Inhibitor Cocktail (CALBIOCHEM #539137 of Thermo Scientific #78430)). The nuclei were harvested at 5000 x g for 7 min in a pre-chilled centrifuge, washed twice with 100 μl of 1.2× restriction NEBuffer 2 (NEB) and resuspended in 200 μl of 1.2× NEBuffer 2. Then, 20% SDS was added to a final concentration of 0.3%, and the nuclei were incubated at 37oC for 1 h with constant shaking. After incubation, 330 μl of 1.2× NEBuffer 2 was added, 20% Triton X-100 was added to a final concentration of 1.8%, and the samples were incubated for 1 h under the same conditions. DNA was digested overnight with 600-800 U of 100 U/μl HindIII-HF restriction endonuclease (NEB) at 37oC with shaking. The next morning, 200 U of HindIII-HF was added, and the samples were incubated for 2 h under the same conditions. The nuclei were harvested for 10 min at 5000 x g and 25oC and resuspended in 50 μl of the collected supernatant. Cohesive DNA ends were biotinylated by adding 7.6 μl of the biotin fill-in mixture prepared in 1× NEBuffer 2 (0.025 mM dATP (Thermo Scientific), 0.025 mM dGTP (Thermo Scientific), 0.025 mM dTTP (Thermo Scientific), 0.025 mM biotin-14-dCTP (Invitrogen), 0.8 U/μl Klenow enzyme (NEB)). The samples were incubated at 37oC for 75 min with slow shaking, and 20% SDS was added to a final concentration of 1.8%; the samples were heated at 65oC for 20 min to inactivate the Klenow enzyme and residual HindIII-HF. The samples were transferred into 10-ml tubes with 3 ml of 1× T4 DNA ligase reaction buffer (Thermo Scientific) supplied with 1.8% Triton X-100, incubated at 37oC for 1 h and then cooled on ice. Next, 100 U of T4 DNA ligase (Thermo Scientific) was added, and the DNA was ligated for 6 h at 22oC with slow agitation. The cross-links were reversed by overnight incubation at 65oC in the presence of proteinase K (100 μg/ml). After cross-link reversal, the DNA was purified by single phenol-chloroform extraction followed by ethanol precipitation (tRNA (Sigma) at a concentration of 7.5 μg/ml and glycogen (Thermo Scientific) at a concentration of 20 μg/ml were used as co-precipitators due to the low amount of DNA in the samples). Biotinylated nucleotides from the non-ligated DNA ends were removed by incubating the Hi-C libraries in the presence of 1.5 U of T4 DNA polymerase (NEB) in NEBuffer 2 supplied with 0.025 mM dATP and 0.025 mM dGTP at 20oC for 4 h. Next, the DNA was purified by phenol-chloroform extraction followed by ethanol precipitation. The DNA pellets were dissolved in 500 μl of sonication buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.1% SDS) and treated with 50 μg of RNase A (Thermo Scientific) for 45 min at 37oC; the DNA was then sheared to a size of approximately 100-1000 bp using a VirSonic 100 (VerTis). The samples were concentrated (and simultaneously purified) using AMICON Ultra Centrifugal Filter Units. The fraction of 100-500-bp DNA fragments was captured using Agencourt AMPure XP beads (Beckman Coulter), and the fragments were eluted with 50 μl of 10 mM Tris-HCl (pH 8.0). The DNA ends were repaired by adding 62.5 μl MQ water, 14 μl of 10× T4 DNA ligase reaction buffer, 3.5 μl of 10 mM dNTP mix, 5 μl of 3 U/μl T4 DNA polymerase (NEB), 5 μl of 10 U/μl T4 polynucleotide kinase (NEB), and 1 μl of 5 U/μl Klenow DNA polymerase (NEB) and incubating at 20oC for 30 min. The DNA was purified with Agencourt AMPure XP beads and eluted with 50 μl of 10 mM Tris-HCl (pH 8.0). To perform an A-tailing reaction, the DNA samples were supplemented with 6 μl 10× NEBuffer 2, 1.2 μl of 10 mM dATP, 1 μl of MQ water and 3.6 μl of 5 U/μl Klenow (exo-) (NEB). The reactions were carried out for 30 min at 37oC in a PCR machine, and the enzyme was then heat-inactivated by incubation at 65oC for 20 min. The DNA was purified using Agencourt AMPure XP beads and eluted with 100 μl of 10 mM Tris-HCl (pH 8.0). Biotin pull-down of the ligation junctions was performed as described previously, with minor modifications. Briefly, 4 μl of MyOne Dynabeads Streptavidin C1 (Invitrogen) beads were used to capture the biotinylated DNA, and the volumes of all buffers were decreased by 4-fold. The washed beads with captured ligation junctions were resuspended in 50 μl of adapter ligation mixture composed of 41.5 μl MQ water, 5 μl 10× T4 DNA ligase reaction buffer (Thermo Scientific), 2.5 μl of Illumina TruSeq adapters and 1 μl of 5 U/μl T4 DNA ligase (Thermo Scientific). Adapter ligation was performed at 22oC for 2.5 h, and the beads were sequentially washed twice with 100 μl of TWB (5 mM Tris-HCl (pH8.0), 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20), once with 100 μl of 1× binding buffer (10 mM Tris-HCl (pH8.0), 1 mM EDTA, 2 M NaCl) and once with 100 μl of CWB (10 mM Tris-HCl (pH 8.0) and 50 mM NaCl) and then resuspended in 20 μl of MQ water. Test PCR reactions containing 4 μl of the streptavidin-bound Hi-C library were performed to determine the optimal number of PCR cycles needed to generate enough PCR.