Fig 2.

Experimental design. (A) Initial transposon mutant libraries are incubated with THP-1 derived macrophages to allow invasion (B) to occur. (C) Extracellular bacteria are killed by the application of lysostaphin. (D) Macrophages are lysed, and surviving bacteria are expanded by growth on solid media (E). (F) To provide a control for outgrowth, initial transposon mutant libraries are incubated in cell culture media and then plated directly on solid media (G). (H) Tn-Seq is performed on the initial library population, the library after macrophage invasion, and the outgrowth control. (I) Tn-Seq output is compared across conditions to identify genes for which insertional inactivation is significantly overrepresented (gene C), underrepresented (gene A), or unchanged (gene B) in mutants successfully invading macrophages relative to counts in initial and/or outgrowth control libraries.