title: GSM1690087: DmBG3-c2_HindIII Hi-C library_rep1; Drosophila melanogaster; OTHER
library_construction_protocol: Hi-C experiment was performed as described previously (Belton et al. 2012) with minor modifications that are described below. 3-5 millions of cells were pelleted and resuspended in 10 ml of 1× PBS (10 mM phosphate buffer, 2.7 mM KCI, 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. Cell suspension was incubated on ice for 5 min, and then cells were pelleted (10 min at 1000 g in pre-chilled (4C) centrifuge). The supernatant was removed, the cell pellet was washed with 500 μl of 1X PBS and then 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-HCI (pH8.0), 150 mM NaCI, 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 g for 7 min in 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. 20% SDS was added to a final concentration of 0.3%, and nuclei were incubated at 37C for 1h with constant shaking. After incubation, 330 μl of 1.2× NEBuffer 2 were added, 20% Triton X-100 was added to a final concentration of 1.8%, and samples were incubated for 1h in the same conditions. DNA was digested overnight with 600-800 U of 100 U/μl HindIII-HF restriction endonuclease (NEB) at 37C with shaking. Next morning, 200 U of HindIII-HF were added and samples were incubated for 2h in the same conditions. Nuclei were harvested at 5000 g for 10 min at 25C 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/ul Klenow enzyme (NEB)). Samples were incubated at 37C for 75 min with slow shaking, then 20% SDS was added to a final concentration of 1.8%, and samples were heated at 65C for 20 min to inactivate 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 37C for 1h 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 22C with slow agitation. The cross-links were reversed by incubation at 65C overnight in the presence of proteinase K (100 ug/ml). After cross-link reversal, DNA was purified by single phenol-chloroform extraction followed by ethanol precipitation (tRNA (Sigma) at a concentration of 7.5 ug/ml and glycogen (Thermo Scientific) at a concentration of 20 ug/ml were used as co-precipitators due to a low amount of DNA in the samples). After precipitation, the pellets was dissolved in 500 μl 10 mM Tris-HCI pH8.0, and DNA was additionally purified to remove residual salts and DTT on AMICON Ultra Centrifugal Filter Units (0.5 ml, 30K, Millipore #UFC5030BK) by washing with 10 mM Tris-HCI pH 8.0 (after two washing steps the final volume of the DNA solution was 40-50 μl). Biotinylated nucleotides from non-ligated DNA ends were removed by incubation of 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 20C for 4 h. Next, 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-HCI (pH 8.0), 10 mM EDTA, 0.1% SDS), the samples were treated with 50 ug of RNAse A (Thermo Scientific) for 45 min at 37C, and DNA was then sheared to the size of approximately 100-1000 bp using a VirSonic 100 (VerTis). Samples were concentrated (and simultaneously purified) on AMICON Ultra Centrifugal Filter Units. Fraction of 100-500 bp DNA fragments was captured by using Agencourt AMPure XP beads (Beckman Coulter), DNA was eluted with 50 μl of 10 mM Tris-HCI (pH 8.0). The DNA ends after the sonication step 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), 1 μl of 5 U/μl Klenow DNA polymerase (NEB) and incubating at 20C for 30 min. DNA was purified with Agencourt AMPure XP beads and eluted with 50 μl of 10 mM Tris-HCI (pH 8.0). To perform an A-tailing reaction, the DNA samples were then supplemented with 6 μl 10X 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 37C in PCR-machine and then their enzyme was heat-inactivated by incubation at 65C for 20 min. DNA was purified using Agencourt AMPure XP beads and eluted with 100 μl of 10 mM Tris-HCI (pH 8.0). Biotin pull-down of the ligation junctions was performed as described previously (Belton et al. 2012) with minor modifications. Briefly, 4 μl of MyOne Dynabeads Streptavidin C1 (Invitrogen) beads were used to capture biotinylated DNA, and the volumes of all buffers were decreased by 4 times. 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 10X 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 22C for 2.5 h. Then 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-HCI (pH8.0), 50 mM NaCI) and then resuspended in 20 μl of MQ water. Test PCR reactions containing 4 μl of streptavidin bound Hi-C library were performed to determine the optimal number of PCR cycles needed to generate enough PCR products for sequencing. PCR reactions (volume of each reaction is 25 μl) 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 98C followed by 6, 9, 12, 15 and 18 cycles of 20 s at 98C, 15 s at 65C, 20 s at 72C. The PCR reactions were run on a 2% agarose gel supplied with ethidium bromide and the number of PCR cycles necessary to obtain sufficient amount of DNA was determined based on visual inspection of gels (typically 12-15 cycles). Four preparative PCR reactions were performed for each sample. PCR mixtures were combined, PCR products were purified with QIAGEN PCR Purification Kit. The DNA was eluted with 50 μl of 10 mM Tris-HCI (pH 8.0) and was run on a 1.8% agarose gel supplied with ethidium bromide. 200-600 bp DNA fragments were cut off the gel, purified on QIAGEN Gel Extraction Kit and sequenced on Illumina HiSeq 2000 by paired-end 101-nt reads.
attributes.tag: GEO Accession
attributes.value: GSM1690087