GSM4708496: RpoN-V5_induction_condition_rep2; Yersinia pseudotuberculosis; ChIP-Seq
Sonicated lysates were purified with antibody. Y. pseudotuberculosis ∆rpoN expressing RpoN::V5 in trans under the inducible araBAD promoter (YPIII, ∆rpoN/pIBX, prpoN::V5 ) as well as Y. pseudotuberculosis expressing RpoN:V5 in cis under its native promoter (YPIII, rpoN::V5/pIBX ) were grown overnight in LB, diluted to OD600 0.05, and grown for 4 h at 26ºC, at which point the prpoN::V5 plasmid was induced with 0.005% arabinose for 30 min. Virulence-induced cultures were shifted to 37ºC after 1 h at 26ºC and incubated for 75 min in calcium-depleted conditions.. Non-tagged WT Y. pseudotuberculosis grown at 26ºC for 4 h was used as a negative control. Cultures were cross-linked with 1% formaldehyde at room temperature for 10 min. For subsequent ChIP-seq, 10 OD600 were used for each biological replicate. Chromatin immunoprecipitation was performed as described previously, with slight modifications (Bonocora et al., 2013). The 1 ml samples were sonicated in AFA milli-tubes using a Covaris E220 sonicator with the following settings: peak power, 140W; duty factor, –5%; cycles/burst, 200; time, 16 min. Immunoprecipitation was done using 90 μL Anti-V5 Agarose Affinity Gel (Sigma A 7345) overnight at 4ºC. ChIP DNA was eluted in 100 μl elution buffer, treated with RNase A and proteinase K, and purified using a ChIP DNA clean & Concentrator Kit (Zymo Research, USA). AMPure XP magnetic beads (Beckman Coulter) 1.5 and 0.8 were used to remove adapters and clean and concentrate the DNA. Failsafe polymerase (Nordic Biolabs AB, Sweden) was used for efficient library amplification.
GEO Accession
GSM4708496