GSM6758208: control, replicate1, Hi-C; Drosophila melanogaster; Hi-C
5–10 million cells were fixed in 1× phosphate-buffered solution (PBS) with 2% formaldehyde for 10 min with occasional mixing. The reaction was stopped by the addition of 2 M glycine to give a final concentration of 125 mM. Cells were centrifuged (1,000 x g, 10 min., 4 °C), resuspended in 50 μl of 1× PBS, snap-frozen in liquid nitrogen, and stored at -80 °C. Defrozen cells were lysed in 1.5 ml isotonic buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% (v/v) NP-40 substitute (Fluka), 1% (v/v) Triton-X100 (Sigma), 1× Halt™ Protease Inhibitor Cocktail (Thermo Scientific) on ice for 15 min. Cells were centrifuged at 2,500 x g for 5 min, resuspended in 100 μl of 1× DpnII buffer (NEB), and pelleted again. The pellet was resuspended in 200 μl of 0.3% SDS in 1.1× DpnII buffer and incubated at 37 °C for 1 hour. Then, 330 μl of 1.1× DpnII buffer and 53 μl of 20% Triton X-100 (Sigma) were added, and the suspension was incubated at 37 °C for 1 hour. Next, 600 U of DpnII enzyme (NEB) were added, and the chromatin was digested overnight (14–16 hours) at 37 °C with shaking (1,400 rpm). On the following day, 200 U of DpnII enzyme were added, and the cells were incubated for an additional 2 hours. DpnII was then inactivated by incubation at 65 °C for 20 min. After DpnII inactivation, the nuclei were harvested for 10 min at 5,000 x g, washed with 100 μl of 1× NEBuffer 2, and resuspended in 50 μl of 1× NEBuffer 2. Cohesive DNA ends were biotinylated by the addition of 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), and 0.8 U/μl Klenow enzyme (NEB)). The samples were incubated at 37 °C for 75 min with shaking (1,400 rpm). Nuclei were centrifuged at 3,000 x g for 5 min, resuspended in 100 μl of 1× T4 DNA ligase buffer (Fermentas), and pelleted again. The pellet was resuspended in 400 μl of 1× T4 DNA ligase buffer, and 75 U of T4 DNA ligase (Fermentas) were added. Chromatin fragments were ligated at 20 °C for 6 hours. The cross-links were reversed by overnight incubation at 65 °C 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 with 20 μg/ml glycogen (Thermo Scientific) as the co-precipitator. After precipitation, the pellets were dissolved in 100 μl 10 mM Tris-HCl pH 8.0. To remove residual RNA, samples were treated with 50 μg of RNase A (Thermo Scientific) for 45 min at 37 °C. To remove residual salts and DTT, the DNA was additionally purified using Agencourt AMPure XP beads (Beckman Coulter). Biotinylated nucleotides from the non-ligated DNA ends were removed by incubating the Hi-C libraries (2 μg) in the presence of 6 U of T4 DNA polymerase (NEB) in NEBuffer 2 supplied with 0.025 mM dATP and 0.025 mM dGTP at 20 °C for 4 hours. Next, the DNA was purified using Agencourt AMPure XP beads. The DNA was dissolved in 500 μl of sonication buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.1% SDS) and sheared to a size of approximately 100–1,000 bp using a VirSonic 100 (VerTis). The samples were concentrated (and simultaneously purified) using AMICON Ultra Centrifugal Filter Units to a total volume of approximately 50 μl. The DNA ends were repaired by adding 62.5 μl MQ water, 14 μl of 10× T4 DNA ligase reaction buffer (Fermentas), 3.5 μl of 10 mM dNTP mix (Fermentas), 5 μl of 3 U/μl T4 DNA polymerase (NEB), 5 μl of 10 U/μl T4 polynucleotide kinase (NEB), 1 μl of 5 U/μl Klenow DNA polymerase (NEB), and then incubating at 20 °C 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 37 °C in a PCR machine, and the enzyme was then heat-inactivated by incubation at 65 °C 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 pulldown 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 comprising 41.5 μl MQ water, 5 μl 10× T4 DNA ligase reaction buffer (Fermentas), 2.5 μl of Illumina TruSeq adapters, and 1 μl of 5 U/μl T4 DNA ligase (Fermentas). Adapter ligation was performed at 22 °C for 2.5 hours, and the beads were sequentially washed twice with 100 μl of TWB (5 mM Tris-HCl (pH 8.0), 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20), once with 100 μl of 1× binding buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 2 M NaCl), 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 required to generate sufficient PCR products for sequencing. The PCR reactions were performed using KAPA High Fidelity DNA Polymerase (KAPA) and Illumina PE1.0 and PE2.0 PCR primers (10 pmol each). The temperature profile was 5 min at 98 °C, followed by 6, 9, 12, 15, and 18 cycles of 20 s at 98 °C, 15 s at 65 °C, and 20 s at 72 °C. The PCR reactions were separated on a 2% agarose gel containing ethidium bromide, and the number of PCR cycles necessary to obtain a sufficient amount of DNA was determined based on the visual inspection of gels (typically 12–15 cycles). Four preparative PCR reactions were performed for each sample. The PCR mixtures were combined, and the products were separated on a 1.8% agarose gel. 200–600 bp DNA fragments were excised from the gel and purified with a QIAGEN Gel Extraction Kit. Two biological replicates were performed.