CarD_plusNB_b
Cereibacter sphaeroides
Protocols: All cells were harvested by high speed centrifugation (approx 5000 xG). Pellets were flash froze in liquid nitrogen before storage at -80 oC. For each biological replicate, a single colony was inoculated into 10 ml M22+ media and grown at 30 degrees Celsius with vigorous shaking for 2-3 days. This 10 ml culture was used to start a larger culture, typically 100 - 200 ml. The larger culture had a starting OD600 of 0.05, and was grown throughout the day at 30 degrees Celsius with shaking until exponential phase, approx 0.5 OD600. At which point novobiocin was added for 20 minutes if required.  When stated, novobiocin was added to a exponentially growing cells (OD600 0.5) to a final concentation of 2 μg/ml Exponentially growing R. sphaeroides, with or without 2 μg/ml novobiocin, were crosslinked with 1% (v/v) formaldehyde for 20 minutes. The reaction was quenched with 0.5 M glycine and cells were resuspended in lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 % (v/v) Triton X-100, 0.1% (v/v) sodium deoxycholate and 1 protease inhibitor cocktail tablet per 50 ml) with 2 mg ml-1 lysozyme. After sonication, 20 μl of chromatin was kept as an input control. Approximately 150 μg of chromatin was immunoprecipitated by the addition of anti-CarD antibodies (a gift from Wilma Ross, University of Wisconsin-Madison) or anti-σ antibodies (Biolegend). Protein-DNA complexes were immunoprecipitated with a 1:1 mix of Protein A/G Dynabeads (Thermo Fisher). Before eluting complexes, beads were washed with lysis buffer, high salt (500 mM NaCl) lysis buffer, wash buffer (20 mM Tris pH 8, 250 mM LiCl, 0.5 % (v/v) NP-40, 0.5 % (v/v) sodium deoxycholate and 1 mM EDTA) and TE. Elution, protein degradation (1 hour incubation with proteinase K at 42 °C) and reverse crosslinking (4 hours at 65 °C) was done in elution buffer (50 mM Tris pH 7.5, 10 mM EDTA and 1 % (v/v) SDS). DNA purified from eluate using a Qiaquick PCR clean up kit (Qiagen).  Sequencing libraries were prepared using the NEB next Ultra II kit (New England Biolabs). Library concentrations were determined by qPCR using a NEB next library quantification kit (New England Biolabs).
ENA first public
2025-10-30
INSDC center name
Institute for Microbiology and Infection, University of Birmingham
INSDC status
public
Submitter Id
E-MTAB-15087:CarD_plusNB_b
broker name
ArrayExpress
collection date
not collected
geographic location (country and/or sea)
not collected
growth condition
M22+ media. 30 degrees Celsius
isolate
not applicable
replicate
biological replicate b
response to treatment
2 μg/ml novobiocin
sample name
E-MTAB-15087:CarD_plusNB_b
scientific_name
Cereibacter sphaeroides
strain
Rhodobacter sphaeroides 2.4.1