Rrp6_I_IP_2
Saccharomyces cerevisiae
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%). To allow the expression of Rho, the yeast cells were grown the same medium but without Doxycycline. Growth was performed during 16 hours incubation at 25°C. All the immunoprecipitated proteins are endogenously tagged (par homologuous recombination in our yeast) by a Myc tag. The antibody used is directed against this MYC tag. We used the anti-cMYC sc-40X from Santa-Cruz Biotechnology. 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
ENA-FIRST-PUBLIC
2019-12-04T04:02:31Z
ENA-LAST-UPDATE
2018-12-19T14:29:48Z
External Id
SAMEA5190600
INSDC center name
CNRS
INSDC first public
2019-12-04T04:02:31Z
INSDC last update
2018-12-19T14:29:48Z
INSDC status
public
Submitter Id
E-MTAB-7539:Rrp6_I_IP_2
broker name
ArrayExpress
common name
baker's yeast
genotype
pCM185-Rho-NLS
growth condition
YNB medium supplemented with SC-TRP and 2% glucose
sample name
E-MTAB-7539:Rrp6_I_IP_2
scientific_name
Saccharomyces cerevisiae
strain
BMA41