title: ChIP-Seq of C. crescentus NA1000: IscR-FLAG, no treatment, replicate 1
design: Cultures from the IscR-FLAG strain were freshly diluted to OD600nm =0.1 in 180 ml PYE at 30C at 200 rpm. After reaching an OD600nm =0.3, cultures continued to grow in the normal condition (PYE medium, n=3) or under iron deficiency condition (PYE with 100 M DP for 2h, n=3). Then, formaldehyde was added to a final concentration of 1% to all cultures, the cells were further incubated at room temperature for 30 minutes, and glycine was added to 0.125 M for 15 min at room temperature. The protocol was followed as described by (Gebhardt et al, 2020; Gozzi et al, 2022). FLAG-tagged IscR was immunoprecipitated using 200 l of Anti-FLAG M2 affinity gel beads (Sigma-Aldrich) and the protein was eluted from the resin in 150 l of buffer. After obtaining the DNA-ChIP fraction, purification was carried out using the Qiaquick PCR purification kit (Qiagen) following the manufacturer's instructions and eluted using 50 l of MilliQ H2O. The purified DNA was used to construct libraries suitable for Illumina sequencing using the NEBNext Ultra II DNA Library Prep Kit for Illumina, and the libraries were sequenced using the NextSeq 500/550 Mid Output Kit v2. 0 (150 cycles) (Illumina) on an Illumina NextSeq 500 (Illumina).