characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: SAMEA2295052
characteristics.characteristic: External Id
characteristics.text: European Molecular Biology Laboratory
characteristics.characteristic: INSDC center name
characteristics.text: 2018-03-08T17:06:56Z
characteristics.characteristic: INSDC last update
characteristics.text: killed
characteristics.characteristic: INSDC status
characteristics.text: ERS388092
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-2180:4C_CRM_2311_WE_3-4h_1
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: fruit fly
characteristics.characteristic: common name
characteristics.text: Protocols: 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-μm Sefar Nitex membrane. The total number of nuclei was estimated by microscopy, and 30 million nuclei aliquots were frozen in liquid nitrogen. 30 million nuclei were resuspended in 500μl 1.2x restriction buffer (60μl 10x DpnII buffer, 440μl H2O). The nuclei were placed at 37C and 0.3% SDS was added before 1h incubation at 37C, shaking gently. 2% Triton X-100 was added to the nuclei before incubating for an additional 1h at 37C, shaking gently. 400U of the DpnII restriction enzyme were added, and the nuclei where incubated overnight at 37C, shaking gently. The next day, 1.6% SDS was added and the nuclei were incubated for 20 min at 65C, shaking gently. The nuclei were transferred in 5ml of 1.4x ligation buffer with 1% Triton X-100 and incubated 1h at 37C, shaking gently. 100U of ligase were added and the reaction was incubated for 4hrs at 16C, followed by 30min at room temperature. The chromatin was de-crosslinked by adding 300μg of proteinase K and incubating overnight at 65C. The next day, 300μg of RNaseA were added to the reaction before 45min incubation at 37C. The DNA was purified by phenol/chloroform followed by ethanol precipitation, and resuspended in 150μl of Tris pH7.5. Libraries were amplified from 320 ng of 4C template using touchdown PCR (94C 2min; 8 cycles of 94C 15sec, 55C 1min, 68C 5sec; 18 cycles of 94C 15sec, 63C 1min, 63C 5sec + 10sec per cycle; 68C 10sec; 22C forever). 100 libraries were multiplexed in equi-molar ratio and sequenced on two HiSeq2000 lanes using 100 bp single-end reads.
characteristics.characteristic: description
characteristics.text: stage 6 to 7 embryo
characteristics.characteristic: developmental stage
characteristics.text: wild type
characteristics.characteristic: genotype
characteristics.text: egg laying
characteristics.characteristic: initial time point
characteristics.text: Drosophila melanogaster
characteristics.characteristic: organism
characteristics.text: Drosophila melanogaster
characteristics.characteristic: organism
characteristics.text: 4C_Growth_Protocol
characteristics.characteristic: organism part
characteristics.text: Drosophila melanogaster
characteristics.characteristic: scientific_name
characteristics.text: mixed sex
characteristics.characteristic: sex
characteristics.text: Twist-H2B
characteristics.characteristic: strain
characteristics.text: 3 to 4 hour
characteristics.characteristic: time
characteristics.text: 4C_CRM_2311_WE_3-4h_1
characteristics.characteristic: title