title: cell culture 5
organism: Escherichia coli str. K-12 substr. MG1655
description: Protocols: All strains used in this study were derived from the E. coli K-12 strain MG1655 derivative NCM3722. The NCM3722:_glnG strain was constructed as described previously (33) .The NCM3722:glnG-3xFLAG strain was constructed by introducing an in-frame fusion encoding three repeats of the FLAG epitope (3x gat tac aag gat gac gat gac aag) to the 3 prime end of glnG. A glnG inducible complementing plasmid (pAPT-glnG-rbs31) was kindly provided by Baojun Wang (34). Bacteria were grown in Gutnick media (33.8 mM KH2PO4, 77.5 mM K2HPO4, 5.74 mM K2SO4, 0.41 mM MgSO4), supplemented with Ho-LE trace elements (35) and 0.4% glucose, and containing either 10 mM NH4Cl (for precultures) or 3 mM NH4Cl (runout experiments) as the sole nitrogen source at 37C, 200rpm. Ammonium concentrations in the media were determined using the Aquaquant ammonium quantification kit (Merck Millipore, U.K.), according to manufacturers instructions. when growth reached the correct stage (N+, OD600nm = 0.3, NRO, growth arrest and N-, 20 min post NRO) samples were taken and cells were subjected in vivo cross-linking with the addition of formaldehyde (Sigma, U.K.) (final concentration 1% (v/v)). For RNAp ChIP cells were subjected to a 15 min treatment with rifampicin (final concentration 150 ug/ml) at 37C prior to cross-linking. Cells were harvested from 20ml of culture by centrifugation, washed twice in Tris-buffered saline (pH 7.5) and pellets were frozen at -80C until required. To fragment the cellular DNA thawed pellets were resuspended in immunoprecipitation (IP) buffer (50 mM HEPES-KOH pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100, 0.1% (w/v) Na deoxycholate, 0.1% (w/v) SDS) supplemented with Complete Protease Inhibitor (Roche, U.K.) before sonication (100% amp, 30 sec pulses for 10 min) (Misonix Ultrasonic Processor S4000), this resulted in fragments of 200-400bp. Cell-debris was removed by centrifugation and the supernatant recovered for IP. A 100 ul aliquot of the supernatant was removed and stored at -20C to act as the input sample which would serve as the background control for ChIP-seq, this sample was de-crosslinked and subjected protein degradation as described for the test samples. Either anti-FLAG (M2) or anti-_ (WP002) were added to the remaining supernatant (approx 750ul) and incubated overnight at 4C on a rotating wheel (a no-antibody control IP was set up at this point also). Sheep anti-Mouse IgG Dynal beads (Invitrogen, U.K.) were prepared by washing 2x with Phosphate-buffered saline (PBS) and 2x with IP buffer, prior to saturating the beads (IP buffer supplemented with Complete protease inhibitor and 1mb/ml BSA) overnight at 4C on a rotating wheel. The blocking solution was removed by applying the beads to a magnet (which was the method used throughout the IP to harvest bead complexes) and removing the supernatant, antibody-nucleoprotein complex was added and incubated for 2 hours at 4C on a rotating wheel. Bead-antibody-nucleoprotein complexes were harvested and subjected to a series of washing steps; 2x IP buffer, 1x IP salt buffer (IP buffer + 0.5 M NaCl), 1x Wash buffer III (10 mM Tris pH 8, 250 mM LiCl, 1 mM EDTA, 0.5% Nonidet-P40, 0.5% (w/v) Na deoxycholate) and a final wash in TE buffer (50 mM Tris, 10 mM EDTA pH 7.5). Immunoprecipitated complexes were then eluted from the beads using an elution buffer (50 mM Tris HCl pH 7.5, 10 mM EDTA, 1% (w/v) SDS) and incubation at 65C for 40 minutes with regular agitation. Beads were removed using the magnet and the eluate carefully removed and diluted 2-fold in nuclease-free water (VWR, U.K.). DNA was purified from the eluate by de-crosslinking and degrading the protein by incubation at 42C for 2 hours and 65C for 6 hours in the presence of Pronase (final concentration 4 mg/ml) prior to Qiagen MiniElute Kit purification (this was carried out for the input sample described above also). DNA was quantified using the high sensitivity dsDNA Qubit assay (Invitrogen, U.K.). Libraries of ChIP-purified DNA were prepared to allow multiplex next generation sequencing using Illumina TruSeq DNA sample preparation kit v2 (Illumina, U.S.A.) according to the manufacturers instructions with the following modifications. 10 ng of ChIP-purified DNA was used to construct each library, barcoded adapters were diluted 10-fold to compensate for the lower DNA amount. An additional 5 cycle PCR was added prior to size selection of libraries to improve yields (PCR completed as described in amplification of libraries in the kit), also an additional gel extraction step was added following final PCR amplification to remove excess primer dimers. PCR amplification for NtrC ChIP libraries was completed using KAPA HiFi HotStart Readymix (Kapa Biosystems, U.S.A.) instead of the kit polymerase as this improved amplification of libraries. Size and purity of DNA was confirmed using high sensitivity DNA analysis kit on an Agilent 2100 Bioanalyser (Agilent, U.S.A.).