Illumina HiSeq 2000 sequencing; ChIP-seq of MG1655 wt, MG1655 ΔphoB, MG1655 Δhns, MG1655 ΔphoB+Δhns and MG1655 PhoB-FLAG strains grown in MOPS media with either low phosphate (0.2 mM) or high phosphate (1.32 mM)
ChIP-seq of MG1655 wt, MG1655 ΔphoB, MG1655 Δhns, MG1655 ΔphoB+Δhns and MG1655 PhoB-FLAG strains grown in MOPS media with either low phosphate (0.2 mM) or high phosphate (1.32 mM)
Subcultures were set up at 1:100 and grown in the same media and conditions as the initial culture to an OD600 of 0.5-0.7.  50 ml of subculture was transferred to a 50 ml conical.   Growth protocol – MOPS-low phosphate:Cultures were started from single colonies in 2 mL MOPS-low phosphate (9.52 mM NH4Cl; 0.523 mM MgCl2; 0.276 mM K2SO4; 0.010 mM FeSO4; 5 x 10-4 mM CaCl2; 50 mM NaCl; 40 mM MOPS; 4 mM Tricine; 0.2 mM K2HPO4) supplemented with 0.4 % glucose at 37°C, shaking at 225 rpm, for ~18 hours.  Subcultures were set up at 1:100 and grown in the same media and conditions as the initial culture to an OD600 of 0.5-0.7. Cells were crosslinked for 20 minutes with formaldehyde (1% final concentration) and then quenched with glycine (0.5 M final concentration).  Cultures were pelleted by centrifugation and washed with Tris-buffered saline (TBS) (same volume as culture volume).  Cell pellets were resuspended in 1 ml TBS, spun for 1 min at max speed in a microcentrifuge and supernatant removed.  Pellets were resuspended in 1 mL FA lysis buffer (50 mM HEPES-KOH, pH 7, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS) with 4 mg/ml lysozyme and incubated at 37°C for 30 minutes. Samples were then chilled and sonicated for 30 minutes in a Bioruptor sonicator (Diagenode) with 30 s on/30 s off pulsing at maximum amplitude. Samples were pelleted in a microcentrifuge to remove debris and supernatants (chromatin) were transferred to a new tube.  FA lysis buffer was added to make 4 x 800 uL aliquots of chromatin.  Samples were stored at -20°C. For the immunoprecipitation (IP) in wild type or FLAG-tagged strains, 800 uL chromatin was incubated with 25 uL Protein A Sepharose slurry (50%) in TBS and 2 uL anti-FLAG (M2) monoclonal antibody (Sigma) for 90 minutes at room temperature with gentle mixing on a rotisserie rotator.  For the immunoprecipitation (IP) in wild type or deletion strains, 800 uL chromatin was incubated with 25 uL Protein A Sepharose slurry (50%) in TBS and 1 uL Sigma 70 antibody for 90 minutes at room temperature with gentle mixing on a rotisserie rotator.  For either IP, following the 90 min incubation, beads were pelleted by centrifugation (1,500 x g for 1 min), supernatant was removed, beads were resuspended in 700 uL FA lysis buffer and transfered to a Spin-X column.  Beads on Spin-X column were washed twice with FA lysis buffer and twice with 10 mM Tris-HCl, pH 7.5 (please note: all wash steps involved incubation of samples with the wash solution for 3 min with gentle mixing on a rotisserie rotator before pelleting beads at 1,500 x g in a microcentrifuge for 1 min).  Beads were resuspended in 100 uL 1X Quick Blunting Buffer (NEB) containing dNTPs (concentration specified by NEB Quick Blunting kit) and 2 uL Quick Blunting Enzyme Mix (NEB), and incubated at room temperature for 30 min with gentle mixing. Beads were washed twice with FA lysis buffer and twice with 10 mM Tris-HCl, pH 8.0. Beads were resuspended in 100 uL 1X Buffer 2 (NEB) containing 2 mM dATP and 2 uL Klenow DNA polymerase (NEB), and incubated for 30 min at 37 degrees Celsius with gentle mixing. Beads were washed twice with FA lysis buffer and twice with 10 mM Tris-HCl, pH 7.5. Illumina adapters were then ligated to the samples using the NEB Quick Ligation kit.  The samples were resuspended in the following mixture:  100uL 1x ligase buffer (50uL 2x buffer + 50uL dH2O), 1 uL NEXTflex ChIP-seq Barcoded adapter (Bio0) and 2 uL Quick DNA Ligase.  The samples were incubated at room temperature for 15 min with gentle mixing.  Beads were washed twice with FA lysis buffer, and once each with high salt FA lysis buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS), ChIP wash buffer (10 mM Tris-HCl, pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5% Nonidet-P40, 0.5% sodium deoxycholate) and TE (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). After the TE wash, the Spin-x column was transferred to a fresh tube, 100uL ChIP elution buffer was added (50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1% SDS) and samples were incubated for 10 minutes at 65 degrees Celsius with occasional agitation. Samples were spun at 1,500 x g for 1 min and eluate was transferred to a fresh tube.  Eluates were decrosslinked by boiling for 10 minutes. DNA was extracted with phenol/chlorofom/isoamyl alcohol, ethanol precipitated, and resuspended in 11 uL H2O. 1 uL DNA was diluted 1:8 uL with sdH20 and 2 uL of this was used for real time PCR amplification (three technical replicates) with oligonucleotides JW3764 (5'AATGATACGGCGACCACCGAGATCTACAC) + JW3765 (CAAGCAGAAGACGGCATACGAGAT ) using an ABI 7500 Fast real time PCR machine. The number of cycles required to reach a Delta Rn score of 0.2 was recorded (“X”). 8 uL of the remaining DNA was amplified using conventional PCR (200uL sample volume divided into 4  x 50 uL reactions) with oligonucleotides JW3764 (100 mM) and JW3765 (100 mM) for X+3 cycles with an annealing temperature of 60 degrees Celsius. PCR products were purified using Ampure XP magnetic beads (Beckman Coulter) (0.8x volume of sample) and resuspended in 15 uL H2O.  Purified PCR products between 200 bp and 600 bp were gel purified from an 8% non-denaturing polyacrylamide gel, eluted overnight at 4°C in 0.4 mL DNA extraction buffer (300mM NaCl, 10mM Tris-Cl pH 8.0, 1mM EDTA pH 8.0) and ethanol precipitated to generate the final ChIP-seq library. The library was resuspended in 12 uL sdH2O and quantified by Qubit.
Experimental Factor: genotype
delta-phoB-delta-hns
Experimental Factor: growth condition
low phosphate
Experimental Factor: immunoprecipitate
sigma 70