title: GSM2028131: DT40_5C_rep2; Gallus gallus; OTHER
library_construction_protocol: Libraries of proximity ligation products were prepared as described previously (Hagege et al. 2007). Briefly, a total of 10 millions cells were pelleted and resuspended in 10 ml of DMEM supplemented with 8% of FBS and 2% of CS 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 1 ml of ice-cold 1× PBS. The cells were lysed for 15 min in 10 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 (Thermo Scientific #78430)). The nuclei were harvested at 1000 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. 20% SDS was added to a final concentration of 1.8% and the samples were heated at 65oC for 20 min to inactivate the restriction enzyme. The samples were transferred into 50-ml tubes with 6.5 ml of 1.07× 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 16oC 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 (glycogen (Thermo Scientific) at a concentration of 20 μg/ml was used as co-precipitator). After precipitation, the pellets were dissolved in 500 μl 10 mM Tris-HCl pH 8.0. To remove residual salts and DTT, the DNA was additionally purified using AMICON Ultra Centrifugal Filter Units (0.5 ml, 30 K, Millipore #UFC5030BK) by washing with 10 mM Tris-HCl pH 8.0 (after two washing steps, the final volume of the DNA solution was 40-50 μl). DNA concentration was measured on Qubit fluorometer (Invitrogen). 5C libraries were prepared in two biological replicates as described previously (Dostie and Dekker, 2007) with minor modifications. Briefly, 5C primer stocks (17 mM) were mixed and diluted in water to a final concentration of 1.7 nM of each primer. 5C primers were mixed with 3C libraries in 10 μl of annealing mixtures of the following composition: 1 μl of annealing buffer (10× NEBuffer 4 (NEB)), 1.7 μl of 5C primer mix, 600 ng of 3C library, 900 ng of salmon testis DNA and MQ water. Samples were denatured for 5 min at 95°C and annealed for 16 h at 57°C. Ligation with 10 U of Taq DNA ligase was performed for 1 h at 57°C. 2 μl of each 5C library was then PCR-amplified individually with primers to T7- and T3-tails using KAPA High Fidelity DNA Polymerase (KAPA). The temperature profile was 5 min at 98oC, followed by 22, 24, 26, 28 and 30 cycles of 20 s at 98oC, 15 s at 65oC, and 20 s at 72oC. The PCR reactions were separated on a 2% agarose gel supplied with 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 28-30 cycles). Four preparative PCR reactions were performed for each sample. The PCR mixtures were combined, and the products were purified with QIAGEN PCR Purification Kit. The DNA was eluted with 50 μl of 10 mM Tris-HCl (pH 8.0) and separated on a 1.8% agarose gel supplied with ethidium bromide; 100-bp fragments were excised from the gel, purified using QIAGEN Gel Extraction Kit and sequenced with an Illumina HiSeq 2000 by single-end 101-nt reads.