DBal_Capture-C_N1pat_4-8h_Rep2
Drosophila melanogaster
Protocols: 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. 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. Three aliquots of 30x106 nuclei in 2 biological replicates of the same nuclei used for the Hi-C experiment were used for each template preparation. The Capture-C libraries were prepared as previously described (Davies et al. 2016), 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.
ENA-FIRST-PUBLIC
2019-04-24T04:04:22Z
ENA-LAST-UPDATE
2018-12-11T09:10:52Z
External Id
SAMEA5163644
INSDC center name
EMBL
INSDC first public
2019-04-24T04:04:22Z
INSDC last update
2018-12-11T09:10:52Z
INSDC status
public
Submitter Id
E-MTAB-7513:DBal_Capture-C_N1pat_4-8h_Rep2
age
4 to 8
broker name
ArrayExpress
common name
fruit fly
developmental stage
stages 8-11
genotype
+/+; +/+ || +/CyO; +/TM3 || +/CyO; +/+ || +/+; +/TM3
initial time point
egg laying
organism part
whole embryo
sample name
E-MTAB-7513:DBal_Capture-C_N1pat_4-8h_Rep2
scientific_name
Drosophila melanogaster
sex
mixed sex
strain
N1pat