4 protocols
growth protocol
Male F1 (+/CyO; +/TM3) and female virginizer flies were crossed in population cages at 25 degr. After three one-hour prelays, embryos were collected at 4-8 h after egg lay. Two independent collections were performed. 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 RT for 15 minutes. The reaction was terminated with 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.
The libraries were sequenced on a HiSeq 2000 (Illumina) in paired end mode.
nucleic acid extraction protocol
1g 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 estimated by microscopy, and 30 million nuclei aliquots were frozen in liquid nitrogen.
nucleic acid library construction protocol
Three aliquots of 30x106 nuclei in 2 biological replicates were used for each 3C template preparation. The Hi-C libraries were prepared as previously described (Sexton et al. 2012), using DpnII as restriction enzyme. The final library was prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina (NEB) according to the manufacturer???s instructions from at least 1 ??g of DNA.