Comparing the chromosome binding profiles of nucleoid structuring protein H-NS in the presence or absence of conditions eliciting the depletion of transcription termination protein NusG in Salmonella
Comparing the chromosome binding profiles of nucleoid structuring protein H-NS in the presence or absence of conditions eliciting the depletion of transcription termination protein NusG in Salmonella
Salmonella bacteria were grown as described in the growth protocol. When the culture reached an OD600 of 0.7-0.8, 30 ml were withdrawn, transferred to 125 ml Erlenmeyer flasks and 1.6 ml of 37% Formaldehyde (Alfa Aesar) were added. The culture was incubated for 30 min at room temperature with gentle agitation. This was followed by the addition of 6.8 ml of a 2.5 M glycine solution and a further 15 min incubation with gentle agitation at room temperature. Cells were centrifuged and the pellet resuspended in 24 ml of TBS buffer (50 mM Tris HCl pH7.4, 150mM NaCl). These steps were repeated once and the cells centrifuged again.  Overnight cultures of Salmonella bacteria (inoculated from single colonies) were diluted 1:100 in LB broth or in LB broth supplemented with 0.1% ARA or 0.1% ARA + 0.4 µg/ml AHTc and grown with shaking (170 rpm) at 37°C to an OD600 of 0.7-0.8.  Bacterial pellets were resuspended in 0.5 ml of Lysis buffer (50mM Tris pH 7.4, 150mM NaCl, 1mM EDTA supplemented with cOmplete™ Mini Protease Inhibitor cocktail (Sigma-Aldrich). Then, 3 µl of Ready-Lyse™ Lysozyme solution, 25 000 – 40 000 U/µl (Lucigen R1804M) were added and incubated on ice for 10 min, followed by the addition of 25 µl of 25% Triton and 450 µl of Lysis buffer. Cells were disrupted in a Covaris 220 focused ultrasonicator (Peak Incidence = 140W; Duty Factor = 5; Cycles per burst = 200). This step was followed by 10 min centrifugation at 16 000 rcf at 4°C. After removing aliquots for “INPUT” controls, supernatants (0.9 ml) were treated overnight with Anti-Flag M2 affinity Gel (Sigma A2220, 40 µl of slurry per sample) on a gyratory wheel at 8°C. Next day, beads were washed in the cold with 500 µl of TBS plus 0.05% Tween 20 for 10 minutes, followed by four TBS washes, of 10 minute each. The elution of 3xFLAG-H-NS DNA complex from the beads was accomplished as follows: 3xFLAG peptide solution (prepared according to Sigma Aldrich instructions) was diluted with TBS (3 µl of 3xFLAG peptide concentrated solution per 100 µl of TBS) and an aliquot of 2.5 times volume (relative to beads) of the peptide-TBS solution was added on top of the beads. Tubes were incubated for 1 hour on the gyratory wheel at 8°C. Samples were centrifuged briefly and supernatant transferred to a clean tube. Inputs as well as samples were treated with Proteinase K (2.5 µl of 2mg/ml solution) at 65°C overnight. DNA was cleaned using Qiagen Mini-elute reaction clean up kit  ChIP DNA fragments were end-repaired and dA-tailed (NEB#E7595), Illumina TruSeq adapters were ligated (NEB#E6040) and libraries were amplified with Kapa Hifi polymerase (Kapa Biosystem #KK2103). Final libraries quality was assessed on an Agilent Bioanalyzer 2100, using an Agilent High Sensitivity DNA Kit.