characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-03-01T00:17:05Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-03-01T00:17:05Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA112145359
characteristics.characteristic: External Id
characteristics.text: CNRS - CBM (UPR 4301)
characteristics.characteristic: INSDC center name
characteristics.text: 2023-03-01T00:17:05Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-03-01T00:17:05Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS14255117
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12378:IP_HPR1moinsRho_1
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: baker's yeast
characteristics.characteristic: common name
characteristics.text: Protocols: S. cerevisiae cells were grown according to standard procedures at 25 °C in synthetic complete medium with bases and amino acids omitted as necessary for selection and with glucose (2%) as a carbon source. The pCM185 vector (CEN, TRP1) from Euroscarf was used as backbone to create the Doxycycline-regulated (Tet off) Rho expression construct (pCM185-Rho-NLS) (Mosrin-Huaman et al., 2009). Strains transformed by Rho expression vector were grown under repressing conditions (5 µg/mL of Doxycycline) at 25°C in YNB medium supplemented with SC-TRP (Synthetic Complete Supplement Mixture from MP Biomedicals) and glucose as carbon source (final concentration 2%). Strains transformed by Rho expression vector were grown under repressing conditions (5 µg/mL of Doxycycline) at 25°C in YNB medium supplemented with SC-TRP (Synthetic Complete Supplement Mixture from MP Biomedicals) and glucose as carbon source (final concentration 2%). DO was adjusting by dilution in order to obtain the same number of cell in each culture/condition. Proteins/DNA crosslinking was performed by adding 1.2 mL of Formaldehyde (37%, SIGMA) to the 45mL of each yeast culture and incubated 20 min at room temperature. Cross-linking reaction was stopped by adding 8mL of Glycine (2.5M) for 5 min and then put on ice. Cells were washed twice with cold TBS and twice in lysis buffer (50 mM Hepes pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate and 1:100 Protease Inhibitor Cocktail from Promega). We then applied a centrifugation step at 13000 RPM, 5min at 5°C. After getting rid of the supernatant, pellet were resuspended in 1mL lysis buffer, and cells were break mechanically with glass beads (add directly to the samples). After recovering 1.2mL (=starting lysate), the cross-linked chromatin was sheared by sonication to reduce average fragment size to ~400 bp. The samples were centrifuged at 2500g and the supernatant recovered. 20 µL of supernatant genomic DNA was used as Input and the remaining was mixed with antibody solution (anti c-MYC sc-40X from Santa Cruz Biotechnology). Tubes were incubated overnight at 4°C, then protein G PLUS-agarose beads (Sigma) were added and rotated for 2 hours at 4°C followed by successive washing steps: 2 times with lysis buffer, 2 times with lysis buffer 2 (similar to lysis buffer but with 360 mM NaCl instead of 140 mM), 1 time with washing buffer and 1 time with TE. DNA was eluted twice at 65°C in elution buffer during 10 minutes. Samples (regardless of Input or IP) were reverse crosslinked at 65°C overnight. Proteinase K digestion of the eluates was performed prior to purification of the genomic DNA samples using the ThermoFisher GeneJET PCR Purification Kit. ChIP libraries preparation was performed following the NEBNext Ultra II DNA Library Prep instructions from NEB
characteristics.characteristic: description
characteristics.text: pCM185-Rho-NLS; Hpr1-Myc
characteristics.characteristic: genotype
characteristics.text: Saccharomyces cerevisiae
characteristics.characteristic: organism
characteristics.text: Saccharomyces cerevisiae
characteristics.characteristic: organism
characteristics.text: 1
characteristics.characteristic: replicate
characteristics.text: Saccharomyces cerevisiae
characteristics.characteristic: scientific_name
characteristics.text: BMA41
characteristics.characteristic: strain
characteristics.text: IP_HPR1moinsRho_1
characteristics.characteristic: title