TS_Capture_TSS_6-8h_Mef2_Rep2_p
Wildtype flies were held in population cages at 25Â°C. After three one-hour pre-lays, embryos were collected at the indicated time (please see ???age???) after egg laying. The collected embryos were dechorionated using 50% bleach and formaldehyde fixed in 10 ml cross-linking solution (50 mM Hepes, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl, 1.8 % formaldehyde, pH 8.0) and 30ml n-heptane on a shaker table at room temperature for 15 minutes. The reaction was terminated with ice-cold 125 mM glycine, 0.1% Triton X-100 in PBS. After washing out the fix, the embryos were blotted dry and frozen in liquid nitrogen. 0.5-1 g of frozen embryos were dounce-homogenized, 20 times with a loose pestle and 10 times with a tight pestle, in 10 ml of chilled HB buffer (15 mM Tris-HCl (pH 7.4), 0.34 M sucrose, 15 mM NaCl, 60 mM KCl, 0.2 mM EDTA and 0.2 mM EGTA) on ice. The lysate was filtered through 2 layers of Miracloth and spun at 3,500g for 10 min to pellet the nuclei. The nuclei were then washed in 10 ml of chilled HB buffer, spun at 3,500g for 10 min and resuspended in 3 ml of PBTB buffer (5% (wt/vol) BSA, 0.1% (vol/vol) Triton X-100 in PBS). The nuclei were dissociated by passing them ten times through a 20-G needle and then ten times through a 22-G needle using a 5-ml syringe, and filtered through a 20-??m Sefar Nitex membrane. The total number of nuclei was determined using a flow cytometer or cell counter.    Sorting of nuclei was performed similar to what has been described in Bonn et al., 2012. Four aliquots of 25 million nuclei were used for preparation of the 3C library for each sample. 3C samples and Capture-C libraries were prepared as previously described (Davies et al. 2016, Ghavi-Helm et al., 2019), using DpnII as restriction enzyme and performing the ligation step"in nucleus. The template was then processed with the Roche Nimblegen SeqCap EZ Library SR system and a double capture strategy.