title: GSM6500793: empty vector ChIP control, replicate 2; Pseudomonas aeruginosa; ChIP-Seq
library_construction_protocol: Chromatin immunoprecipitation (ChIP) protocol was based on the method described earlier (Kawalek et al, 2018; Nucleic Acids Res. 46:9, 4592-4606; Modrzejewska et al, 2021; mSystems 6:4, e0001521). Two cultures of each strain with similar OD600 were combined, formaldehyde was added from a 37% stock to a final concentration of 1%, and the cultures were kept for 10 min at room temperature with gentle inverting. Glycine was added to a final concentration of 0.5 M and the cultures were incubated for 5 min at room temperature with gentle rotation. Cells were then harvested by centrifugation (20 min, 3000 x g, 25 mL of culture in 50 mL conical tubes, 4 tubes per strain), washed twice with ice-cold Tris-buffered saline (25 mM Tris-HCl, 150 mM NaCl, pH=7.6), resuspended in 1 mL of TBS, transferred to a fresh 1.5 mL tube and centrifuged (2 min, 6000 x g). The cell pellets were frozen at -70oC. The pellets were thawed on ice, suspended in 0.5 mL of lysis buffer [10 mM Tris-HCl, 20% (w/v) sucrose, 50 mM NaCl, 10 mM ethylenediaminetetraacetic acid (EDTA), pH=8.0] supplemented with 10 µl of Protease Inhibitor Cocktail (Sigma, PA8849) and 125 µL of lysozyme (20 mg/mL) and incubated for 30 min at 37oC. Four samples for each strain were combined in 15 mL. Subsequently, 1.5 mL of IP-buffer [50 mM Tris-HCl, pH=7.5, 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100, 0.1% (m/v) sodium deoxycholate], 30 µL protease inhibitor cocktail, PMSF (phenylmethanesulfonyl fluoride) to a final concentration of 2 mM, SDS (sodium dodecyl sulphate) to a final concentration of 0.1% were added to each sample. The samples were sonicated 20 times for 20 s each with 1 min break on ice using a Microson ultrasonic cell disruptor Xl2000 (Misonix) with 5-mm tip and 90% of output power to obtain DNA fragments of an average length of 250-350 bp. DNA fragmentation was checked by agarose gel electrophoresis. After sonication, samples were aliquoted (1 mL in 1.5 mL tubes) and stored at -70oC. Sonicated lysate was thawed on ice and 150 μL of each strain variant was incubated with 20 μL of magnetic beads coupled with protein A (Dynabeads Protein A, Invitrogen, 10001D) previously separated from original suspension using Magnetic Separation Stand. Such pre-clearing step was performed for minimum 1 hour at 4oC with rotation of the mixtures. In the meantime 50 μL of magnetic beads were prepared for each reaction by separating from the suspension as above. It was then mixed with anti-FLAG mouse antibodies (1-10 μg per reaction, 4 μL for this procedure) [DYKDDDDK Tag polyclonal  antibodies; 1 mg/mL; PA1-985B; Invitrogen] diluted in 200 μL of PBS with 0.05% Tween-20. Mixtures of magnetic beads and antibodies were incubated for 10 minutes at 4oC with gentle rotation. Beads with bound antibodies were then separated from the supernatant, washed once with 200 μL of the PBS with 0.05% Tween-20 solution and stored on ice. Pre-cleared lysate was separated from the beads used for pre-incubation and added to the beads coated with antibodies. Mixture containing lysate and magnetic beads with antibodies was incubated at 4oC for 20 minutes with mixing on rotator. Beads were then collected and washed. Elution was performed twice for 15 minutes in 50 μL at 65oC in thermoblock with shaking (1400 rpm). All 100 μL of obtained eluates were incubated with 1 μL of RNase A (100 mg/mL, Qiagen, 19101) for 30 min at 65oC. Then, 5 μL of Proteinase K (20 mg/mL, Qiagen, 19133) was added and the samples were incubated for 1 h at 50oC followed by overnight incubation at 65oC. Such prepared probes were stored on ice and 3 μL of 3 M sodium acetate (pH=5) was added. DNA purification was performed using Qiaquick Qiagen PCR purification Kit according to manufacturer's instructions. The DNA was stored at -20oC. NGS library was constructed using QiaSeq Ultralow Input Library kit. Sample was further quality checked on 1% agarose gel and concentration was measured using qPCR KAPA Library Quantification Kit.