GSM4476156: Total-Input_C.crescentus; Caulobacter vibrioides NA1000; ChIP-Seq
Cells were lysed in a Ready-Lyse lysozyme solution (Epicentre Technologies) according to manufacturer's instructions and lysates were sonicated (Bioruptor® Pico) in an ice-water bath for 15 cycles 30 seconds ON and 30 seconds OFF, to shear DNA fragments to an average length of 0.3-0.5 kbp and cleared by centrifugation at 14,000 g for 2 minutes at 4°C. The volume of the lysates was then adjusted (relative to the protein concentration) to 1 mL using ChIP buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl (pH 8.1), 167 mM NaCl plus protease inhibitors (Roche, Switzerland) and pre-cleared with 80 μL of protein-A (rabbit antibodies) or –G (mouse antibodies) agarose (Roche) and 100 μg BSA. Ten percent of the supernatant was removed and used as total chromatin input DNA as described before (Delaby et al., 2019).Two microliters of polyclonal antibodies to TrcR, or of a mix of RNA polymerase from a monoclonal antibody sampler kit (ratio 1:1:1:1, Biolegend), or of monoclonal antibodies to σ70 (BioLegend, Mouse IgG2b) were added to the remains of the supernatant, incubated overnight at 4 °C with 80 μL of protein-A or -G agarose beads pre-saturated with BSA, washed once with low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1) and 150 mM NaCl), high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1) and 500 mM NaCl) and LiCl buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA and 10 mM Tris-HCl (pH 8.1)), and twice with TE buffer (10 mM Tris-HCl (pH 8.1) and 1 mM EDTA). The protein DNA complexes were eluted in 500 μL freshly prepared elution buffer (1% SDS and 0.1 M NaHCO3), supplemented with NaCl to a ﬁnal concentration of 300 mM and incubated overnight at 65 °C to reverse the crosslinks. The samples were treated with 2 μg of Proteinase K for 2 h at 45 °C in 40 mM EDTA and 40 mM Tris-HCl (pH 6.5). DNA was extracted using phenol:chloroform:isoamyl alcohol (25:24:1), ethanol precipitated using 20 μg of glycogen as carrier and resuspended in 100 μL of water. Immunoprecipitated chromatin was used to prepare sample libraries used for deep-sequencing at Fasteris SA (Geneva, Switzerland). ChIP-Seq libraries were prepared using the DNA Sample Prep Kit (Illumina) following manufacturer's instructions.