characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-06-30T00:24:13Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-06-30T00:24:13Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA112361279
characteristics.characteristic: External Id
characteristics.text: EMBL
characteristics.characteristic: INSDC center name
characteristics.text: 2023-06-30T00:24:13Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-06-30T00:24:13Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS14471616
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12639:WE_2-3h_K27ac_rep1
characteristics.characteristic: Submitter Id
characteristics.text: 2 to 3
characteristics.unit: hour
characteristics.characteristic: age
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: fruit fly
characteristics.characteristic: common name
characteristics.text: Protocols: Wildtype Drosophila melanogaster (Oregon R) flies were grown from eggs in population cages at 25 °C. 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. 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 : The nuclei were resuspended in 1 ml of ice-cold nuclear lysis buffer (50 mM HEPES pH 8.0, 10mM EDTA, 0.5% N-Laurylsarcosine supplemented with Complete Protease Inhibitor, Roche), and incubated on ice for 20 minutes. Nuclei were sonicated using a Bioruptor (Diagenode) to generate 250-500 bp chromatin fragments. The chromatin was centrifuged at 16,000xg and the supernatant containing the chromatin was aliquoted into fresh tubes and stored at -80°C until use. The quality of the sheared chromatin was determined by agarose gel electrophoresis to observe chromatin fragment size distribution. Chromation IP Step: For ChIP-seq, antibodies were incubated overnight with up to 2.5 µg chromatin in RIPA buffer (140mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% Triton-X-100, 0.1% SDS, 0.1% sodium deoxycholate, supplemented with Complete Protease Inhibitor, Roche) in a total volume of 900 µl. (We used 2.5 µg chromatin for incubations with anti-CTCF antibody, 2 µg for anti-Su(Hw), 1 µg for anti-BEAF and 2 µg for H3K27ac.) The next day 25 µl of magnetic protein A/G beads (Dynabeads, Invitrogen, 10002D and 10004D) were washed with 1 ml of RIPA buffer and added to the IPs for an additional 3 hour incubation on a rotating wheel at 4¡C. For the BEAF-32 ChIP, 25 µl of protein G beads were combined with 100 µl of the BEAF-32 antibody (DSHB, #1553420) and 300 µl RIPA buffer for 2 hrs. This was followed by two washes with RIPA and resuspension in 100 µl of RIPA buffer, which was added to the purified chromatin and incubated on a rotating wheel at 4¡C over night. The ChIPs were then washed for 10 min on a rotating wheel: once with 1 ml RIPA buffer, 4 times with 1 ml RIPA-500 (500 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, supplemented with Complete Protease Inhibitor, Roche), once with 1 ml LiCl buffer (250 mM LiCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5% IGEPAL CA-630, 0.5 % sodium deoxycholate) and twice with 1 ml TE buffer (10 mM Tris pH 8.0, 1 mM EDTA) on a magnetic rack in the cold room. The chromatin was then RNase-treated (#10109142001, Roche), reverse cross-linked overnight with 0.5 mg/ml Proteinase K and 0.5% SDS at 65°C. The next day the DNA was purified using Phenol-Chloroform isolation and precipitated with ethanol, sodium acetate pH 5.3 and glycogen to obtain pure DNA. For ChIP NGS library prep the following kit was used: NEBNext Ultra II DNA Library Prep Kit for Illumina E7645S.
characteristics.characteristic: description
characteristics.text: embryo stage
characteristics.characteristic: developmental stage
characteristics.text: wild type genotype
characteristics.characteristic: genotype
characteristics.text: not applicable
characteristics.characteristic: isolate
characteristics.text: Drosophila melanogaster
characteristics.characteristic: organism
characteristics.text: Drosophila melanogaster
characteristics.characteristic: organism
characteristics.text: whole embryo
characteristics.characteristic: organism part
characteristics.text: Drosophila melanogaster
characteristics.characteristic: scientific_name
characteristics.text: mixed
characteristics.characteristic: sex
characteristics.text: Oregon-R
characteristics.characteristic: strain
characteristics.text: WE_2-3h_K27ac_rep1
characteristics.characteristic: title