characteristics: ERC000011
characteristics: ENA-CHECKLIST
characteristics: 2022-11-01T00:15:33Z
characteristics: ENA-FIRST-PUBLIC
characteristics: 2022-11-01T00:15:33Z
characteristics: ENA-LAST-UPDATE
characteristics: SAMEA110321372
characteristics: External Id
characteristics: EMBL
characteristics: INSDC center name
characteristics: 2022-11-01T00:15:33Z
characteristics: INSDC first public
characteristics: 2022-11-01T00:15:33Z
characteristics: INSDC last update
characteristics: public
characteristics: INSDC status
characteristics: ERS12418980
characteristics: SRA accession
characteristics: E-MTAB-91561657524261:GAF_NC14_rep2
characteristics: Submitter Id
characteristics: 2h10-2h40
characteristics: age
characteristics: ArrayExpress
characteristics: broker name
characteristics: fruit fly
characteristics: common name
characteristics: Protocols: Wildtype Drosophila melanogaster (Oregon R) flies were grown in population cages at 25 degr. Independent collections of staged embryos were performed for each biological replicate. 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. Specifically for CP190 ChIPs, the same collection procedure was used, but using a final 3.0 % concentration of formaldehyde, for 30 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. A small number of embryos from each collections was set aside to stage each collection. Chromatin Preparation Step : Nuclei were extracted from the frozen embryos by Dounce homogenization and differential centrifugation (Cell lysis buffer: 5mM Hepes, pH 8, 85mM KCl, 0.5% NP40 + protease inhibitors). The nuclei were lyzed in 50mM Hepes, pH 8, 10mM EDTA?Na2, 0.5% N-Laurylsarkosin + protease inhibitors at RT for 20 min and the chromatin was sheared to ca. 250 bp fragments using a Bioruptor sonicator waterbath (Diagenode). After clearing the chromatin by centrifugation (20.000g at 4 degr for 10 min), aliquots were frozen at -80 degr. Chromation IP Step: 5 micrograms of chromatin were mixed with TE and cold IP dilution buffer. 1% Input was isolated at this step. The chromatin was incubated with the appropriate antibody overnight on a rotating wheel. Then, the antibody/antigen complexes were incubated with Protein A/G Beads, followed by multiple washes. The DNA was eluted after RNAase treatment, protease digestion and phenol chloroform extraction. For ChIP NGS library prep the following kit was used: NEBNext Ultra II DNA Library Prep Kit for Illumina E7645S
characteristics: description
characteristics: NC14
characteristics: developmental stage
characteristics: wild type genotype
characteristics: genotype
characteristics: egg laying
characteristics: initial time point
characteristics: Drosophila melanogaster
characteristics: organism
characteristics: Drosophila melanogaster
characteristics: organism
characteristics: whole organism
characteristics: organism part
characteristics: Drosophila melanogaster
characteristics: scientific_name
characteristics: mixed
characteristics: sex
characteristics: OregonR
characteristics: strain
characteristics: GAF_NC14_rep2
characteristics: title