design: ChIP-seq experiment: DnaA binding to E. coli chromosome before stress, during stress, and stress recovery
library_construction_protocol: Amino acid starvation was induced using serine hydroxamate (SHX) treatment. E. coli cells were grown in LB medium at 30°C with aeration until the optical density at 600 nm (OD600) reached 0.6 (before stress sample). SHX was added at a final concentration of 4 mM, and the cultures were incubated for an additional 2 h (stress sample). Cells were harvested by centrifugation, washed twice in LB medium to remove residual SHX, and resuspended in fresh LB medium. Recovery was monitored by growing the cells for 30 in LB medium (recovery sample). 40 ml of bacterial cultures (before stress, during stress or recovery) were treated with 1% formaldehyde for 20 minutes at room temperature. Crosslinking was stopped by adding glycine at final concentration 500 mM and incubating for 5 minutes. The cultures were then centrifuged and washed with TBS. Following crosslinking, cells were lysed by lysozyme treatment and sonication. A 2% aliquot of each lysate was used as the input DNA controls (input samples). Immunoprecipitation was performed using anti-DnaA rabbit antibodies and Protein A Sepharose beads pre-blocked with a blocking buffer (0.2% BSA, 1 mg/mL PVP, 1×TBS). The immunoprecipitated DNA and input DNA samples were decrosslinked and purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel). Purified DNA was subsequently analyzed using Next-Generation Sequencing (ChIP-seq). ChIP-seq were performed in three biological replicates for each condition. Library preparation was performed by Novogene (United Kingdom)