Mycobacterium tuberculosis
Protocols: M. tuberculosis H37Rv delta-glnR were used in this study. M. tuberculosis was grown aerobically in Middlebrook 7H9 liquid broth (supplemented with 0.2% glycerol, 0.025% Tyloxapol and 10% OADC) at 37degrees C, 100 rpm. Optimised nitrogen limiting conditions have been described {Behrends:2012dw, Jenkins:2012jh}. Briefly, a 10 day culture of M. tuberculosis was washed twice in nitrogen free Sauton's medium (0.05% (w/v) KH2PO4, 0.05% (w/v) MgSO4, 0.2% (w/v) citric acid, 0.005% (w/v) ferric citrate, 0.2% (v/v) glycerol, 0.0001% (v/v) ZnSO4, 0.015% (v/v) Tyloxapol) and inoculated into Sauton's nitrogen free medium, supplemented with 30 mM (nitrogen excess) ammonium chloride (Ultra pure; Sigma) to a starting OD60nm0 of 0.04. Growth was monitored by OD600nm. Ammonium ions in the culture supernatant were quantified using an AquaQuant Ammonium detection kit (Merck). The M. tuberculosis delta-glnR mutant was constructed by a recombineering approach as previously described {vanKessel:2007hv} replacing the entire glnR gene with a hygromycin resistance cassette {Jenkins:2012jh}. Primers used to amplify the flanking regions and to confirm mutant construction are given in Table S1. Successful construction of the mutant was confirmed by flanking PCR and Western analysis for the GlnR protein using a GlnR specific antibody previously described {Jenkins:2013gh} Purified rabbit anti-GlnR polyclonal antibody was added to the sonicated cell extract and incubated overnight at 4 degrees C. Sheep anti-rabbit IgG Dynal beads (Invitrogen) were prepared by washing 2x PBS and 2x IP buffer, before bead saturation overnight in blocking solution (IP buffer, EDTA-free protease inhibitor tablet, 1 mg/ml BSA). Blocking solution was removed and bead-sonicated sample complex incubated for 3 hours at 4 degrees C. To harvest the bead-antibody-DNA complex a magnet was used. The complex was then subject to a series of washing steps; 2x IP buffer, IP buffer plus 500 mM NaCl, wash II (10 mM Tris pH 8, 250 mM LiCl, 1 mM EDTA, 0.5% Nonidet-P40, 0.5% (w/v) Na deoxycholate), TE buffer (50 mM Tris, 10 mM EDTA pH 7.5). Elution of DNA was performed by addition of elution buffer (50 mM Tris.HCl pH 7.5, 10 mM EDTA, 1% (w/v) SDS) and incubation at 65 degrees C for 40 min. Beads were separated by magnetism and the supernatant harvested. Elucidate was diluted 2-fold in nuclease free H2O (Qiagen), followed by protein degradation with the addition of 4 mg/ml Pronase and incubated: 42 degrees C for 2 hours and 65 degrees C for 6 hours. DNA was subsequently purified using the Qiagen MiniElute kit and DNA quantified using the dsDNA Qubit (Invitrogen). DNA was prepared for next generation sequencing using the Illumina ChIP-seq DNA sample preparation kit according to the manufacturer's protocol, with the addition of a second gel extraction step after PCR amplification, to remove excess primer dimers. DNA size and purity was confirmed by DNA Bioanalyser (Agilent).
ENA checklist
ERC000011
INSDC center alias
Imperial College
INSDC center name
Imperial College
INSDC first public
2014-11-30T17:02:01Z
INSDC last update
2014-04-25T16:19:27Z
INSDC status
public
SRA accession
ERS445121
Sample Name
ERS445121
alias
E-MTAB-2492:sample_1
broker name
ArrayExpress
organism
Mycobacterium tuberculosis
phenotype
delta-glnR
strain
H37Rv (ATCC 27294)
title
sample_1