GSM4946270: Wpl1-AID, thiolutin [KJ5]; Saccharomyces cerevisiae; Hi-C
Our Hi-C protocol was adapted for S. cerevisiae from ref. (Rao, S. S. et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159, 1665-1680, doi:10.1016/j.cell.2014.11.021 (2014)). Fifty ml of cell culture (at an OD of 1.0) was fixed with 3 % formaldehyde for 20 minutes at 30°C, before the reaction was quenched by adding glycine to 0.125 M final concentration for 5 minutes at room temperature. Cells were washed once with 1 x PBS, before being resuspended in 5 ml pre-spheroplasting buffer (100mM PIPES (pH 9.4), 10mM DTT). The cells were incubated 5 minutes at room temperature and then pelleted (1500 g for 5 minutes at room temperature), before being resuspended in 5 ml spheroplasting buffer (50mM KH2PO4/K2HPO4 (pH 7.5), 1 M Sorbitol, 10 mM DTT). Twenty-five μl of 10 mg ml-1 100T Zymolyase (Nacalai Tesque, 07665-55) was added and cells were incubated for 15 minutes at 30°C. The spheroplasts were pelleted (500 g for 5 minutes at 4°C) and washed twice with ice-cold spheroplasting buffer (containing only 1 mM DTT). The spheroplasts were then resuspended in 250 μl of ice-cold Hi-C lysis buffer (10 mM Tris-HCl (pH 8.0), 10 mM NaCl, 0.2 % Igepal CA630) supplemented with 50 μl 6 x Complete (Roche) and 3 μl protease inhibitor (Sigma, P8215), and incubated on ice for 15 minutes. The spheroplasts were pelleted (1500 g for 5 minutes at 4°C) and washed twice with 500 μl of ice-cold Hi-C lysis buffer. After the last wash step the supernatant was discarded and the spheroplasts were incubated for 6 minutes at 62°C. Thereafter sodium dodecyl sulphate (SDS) was added to a final concentration of 0.2 % and the reaction was immediately and thoroughly mixed by inversion, and thereafter incubated at 62°C for 10 minutes. After addition of 80 μl H2O, 25 μl 10 % Triton X-100 was added to quench the SDS. The reaction was mixed by inversion and incubated at 37°C for 15 minutes. Thereafter, 28 μl 10 x NEB DpnII buffer and 500 units of DpnII (NEB, R0543M) was added, and the chromatin was digested overnight at 37°C. At the end of the incubation, the reaction was supplemented with 250 units of DpnII and incubated for 1 hour at 37°C. Then, the restriction enzyme was inactivated at 62°C for 20 minutes. The presence of intact and individual DNA masses throughout the spheroplasting, digestion and ligation steps was confirmed by DAPI (4′,6-diamidino-2-phenylindole)-staining and microscopy. Marking and repairing DNA ends, proximity ligation, crosslink reversal, DNA shearing, size selection, biotin pull-down, preparation for Illumina sequencing, final amplification (15 cycles) and purification was performed as in ref. (Rao, S. S. et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159, 1665-1680, doi:10.1016/j.cell.2014.11.021 (2014)).
GEO Accession
GSM4946270