characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-08-01T00:16:44Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-08-01T00:16:44Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA111346081
characteristics.characteristic: External Id
characteristics.text: Institut de Genomique Fonctionnelle de Lyon
characteristics.characteristic: INSDC center name
characteristics.text: 2023-08-01T00:16:44Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-08-01T00:16:44Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS13443634
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12153:7kb_2-5h_twi2_rep1
characteristics.characteristic: Submitter Id
characteristics.text: 2 to 5
characteristics.unit: hour
characteristics.characteristic: age
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: fruit fly
characteristics.characteristic: common name
characteristics.text: Protocols: Freshly hatched adults of the appropriate genotype were placed in embryo collection vials with standard apple cap plates. Drosophila embryos were collected on apple juice agar plates at 25 °C at the appropriate time-point (after 3 pre-lays of 1 hour for stage-specific collections), dechorionated using 50% bleach, and washed alternately with water and PBS + 0.1% Triton X-100. The embryos were covalently crosslinked in 1.8% formaldehyde for 15 min at room temperature and stored at -80°C. About 100 to 1000 frozen embryos were homogenized in 10 ml of chilled cell lysis buffer (10mM Tris-HCl, pH8; 10 m m NaCl; 0.2% IGEPAL + Complete) on ice. The lysate was incubated 15 min on ice, and spun at 3,000g for 5 min to pellet the nuclei. The nuclei were then washed in cell lysis buffer, spun at 3,000g for 5 min and resuspended in 50 µl of 0.3% SDS before 10 min incubation at 37C. 148µl of water and 25µl of 10% Triton X-100 were added to the nuclei before incubating for an additional 15 min at 37C. 25µl of 10x CutSmart buffer and 25U of the MboI restriction enzyme were added, and the nuclei were incubated for 90 min at 37C, shaking gently. Another 25U of the MboI were added and the nuclei incubated for another 90 min. The nuclei were then incubated for 20 min at 62C to inactivate the enzyme. The following components were added before incubating for 2 hours at 25C with gently shaking: 556µl of water, 100µl of 10x ligation buffer, 80µ of 10% Triton, 12µl of 10mg/ml BSA, and 2000U of T4 DNA ligase. Another 2000U of T4 DNA ligase was added and the nuclei incubated for another 2 hours. The nuclei were spun at 2500g for 5min and the pellet ressuspended in elution buffer (10mM Tris-HCl, pH8; 1 mM EDTA; 0.5M NaCl; 1% SDS solution; 2µg/µl proteinase K) and incubated 30 min at 55C then overnight at 65C. The next day, 100μg of RNaseA were added to the reaction before 15min incubation at 37C. The DNA was purified using the Zymo ChIP DNA Clean & Concentrator kit and elute in 50 µL of 10 mM Tris-Cl pH 8.0. The DNA was then digested with NlaIII overnight at 37C. The next day, the sample was incubated for 20 min at 65C to inactivate the enzyme. The second liation was carried out using 200U of T4 DNA ligase for 4 hours at 16C then 30 min at room temperature. The sample was purified using Nucleomag PCR beads and eluted in 300µl of 10 mM Tris-HCl pH 8.0. The DNA was amplified from 320 ng of 4C template for 2 cycles using the Q5 Hot Start DNA Polymerase. The PCR product was purified using SPRIselect beads (Beckman Coulter) and 100 ng of each PCR product was used to generate the final libraries using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs). The libraries were indexed for multiplexing using NEBNext multiplex oligos kit for Illumina (New England Biolabs).
characteristics.characteristic: description
characteristics.text: embryonic stage 5 to 9
characteristics.characteristic: developmental stage
characteristics.text: E3(+7.5kb)
characteristics.characteristic: genotype
characteristics.text: pool of 100 to 1000 embryos
characteristics.characteristic: individual
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: 1
characteristics.characteristic: replicate
characteristics.text: Drosophila melanogaster
characteristics.characteristic: scientific_name
characteristics.text: mixed
characteristics.characteristic: sex
characteristics.text: E3(+7.5kb)
characteristics.characteristic: strain
characteristics.text: 7kb_2-5h_twi2_rep1
characteristics.characteristic: title
characteristics.text: Twi2
characteristics.characteristic: viewpoint