GSM4185698: Repli_Late_1; Caenorhabditis elegans; OTHER
For ChEC: Worm cultures were synchronized by embryo isolation with sodium hypochlorite for 4 generations. Embryos were treated with chitinase (1h, RT , 2 U/ml), washed twice in buffer A (15 mM Tris pH 7.5, 0.1 mM EGTA, 0.34 M sucrose, 0.2 mM spermine, 0.5 mM spermidine, 0.5 mM PMSF) and then resuspend in buffer A supplemented with detergents (0.25% NP-40 and 0.1% Tx-100). Nuclei were released with a glass dounce homogenizer and 15 stokes using the loose inserting pestle and 15 stokes using the tight inserting pestle. Debris were spun down at 100 g for 2 minutes and the supernatant, containing nuclei, was saved. The debris were resuspended in buffer A supplemented with detergents and the nuclei isolation step was repeated. The supernatants containing the nuclei were pooled, and the nuclei were pelleted at 1000 g for 10 min. Nuclei were washed once in buffer A, once in buffer B (15 mM Tris pH 7.5, 80 mM KCl, 0.1 mM EGTA, 0.2 mM spermine, 0.5 mM spermidine, 1 mM PMSF), and resuspended in buffer B. MNase was activated by addition of CaCl2 to a final concentration of 2 mM at 30°C. Samples were incubated for 5 to 10 minutes (for CDT-1 ChEC), 5 to 7 minutes (for TRES-1 ChEC) or for 1 h (free MNase control). MNase was inactivated by adding an equal volume of 2x Stop buffer (400 mM NaCl, 20 mM EGTA). Samples were treated overnight with Proteinase K and SDS at 55°C. Phenol-chloroform-isoamyl treatment was used to remove Proteinase K prior the RNase treatment (Roche, Cat. No. 11119915001) for 1h30 minutes at 37°C. After chloroform extraction, DNA was precipitated overnight with ethanol and NaCl. DNA was resuspended overnight in TE prior to constructing the libraries. For EdU-seq and repli-seq: Cells were extracted from embryos as described in (Bianchi and Driscoll, 2006). Briefly, embryos were isolated using sodium hypochlorite, treated with chitinase (1h, RT, 2U/ml), washed once in M9 and resuspend in 10ml of L-15 medium (supplemented with 50 ml FBS (heat inactivated, Gibco), 7.7 g sucrose and 5 ml pen/strep (1:100)). Cells were dissociated with three passes through a syringe with a 22 gauge needle and two passes through a syringe with 26 gauge needle. Debris were pelleted by spinning at 80 g for 1 min, and the supernatant, containing cells, was saved. Debris were resuspended in supplemented L-15 medium and cell extraction was repeated twice more. Supernatants were pooled together and filtered using 5 micron filters. Cells were pelleted at 500 g for 15 min and resuspend in supplemented L-15 medium. Libraries were constructed using NEBNext® Ultra™ II DNA Library Prep NEBNext® Ultra™ II DNA Library Prep