ChIP-qPCR Protocol for Lsr2 Binding in Mycobacteria

For each ChIP library, 50 ml of Mabs-S-Δlsr2-Clsr2-FLAG was grown until an OD₆₀₀ of 0.5 and fixed in 1% formaldehyde (Euromedex) and lysed using a Precellys grinder (3 cycles: 6,700 rpm −3 × 20 s ON/60 s OFF, Bertin Technologies) and VK05 beads. The bacterial chromatin was sheared in 100–300 bp fragments using a Bioruptor Pico (Diagenode). Chromatin Immunoprecipitation was performed as described previously (Grainger et al., 2006 (link)) using IgG M2 anti-FLAG (Sigma, F1804) and Anti-GFP (G6539, Sigma) mouse monoclonal antibodies attached to proteins A/G coupled to magnetic beads (Thermo fisher). The immunoprecipitated DNA and 1% of the total input were reverse-crosslinked and eluted using the iPure v2 kit (Diagenode). The quantitative PCR tests were performed using the SsoFast evergreen supermix (Bio-Rad). Enrichment of Lsr2 binding at the GPL operon operator was calculated using the “Percent Input” method using the primers MAB_4100c_qPCR1 and MAB_4100c_qPCR3 (Supplementary Table S1).