Elucidating transcription factor binding dynamics in Salmonella Typhimurium under macrophages in vivo conditions using a low-Input ChIP-exo approach [ChIP-mini]
We present the low-input ChIP-exo minimization (ChIP-mini) method, which is suitable for processing small bacterial cell numbers, as few as 4.8x106 (bacterial culture volume: 10 µl). This method faithfully maintains the relevant binding profiles of Escherichia coli K-12 MG1655 RpoD, as generated by the traditional ChIP-exo method (bacterial culture volume: 50 ml). Additionally, ChIP-mini was applied in the conventional infection process of mouse macrophages-like cells (J774A.1, RAW264.7) and human epithelial cells (HeLa, INT407) with Salmonella Typhimurium 14028s to directly capture both host extracellular and intracellular bacteria (extracellular and intracellular ChIP-mini). These applications of ChIP-mini in infectious S. Typhimurium provided near-base pair resolution to measure the impact of environmental changes on the binding profiles of H-NS and RpoD. Overall design: [Intracellular ChIP-mini] Mouse macrophage-like cells (J774A.1, RAW 264.7) and human epithelial cells (HeLa, INT407) intracellular S. Typhimurium 14028s WT and hns-8myc tagged cells were co-crosslinked for the intracellular ChIP-mini with a biological replicate. [IMS intracellular ChIP-mini] Mouse macrophage-like cells (J774A.1) intracellular S. Typhimuirum 14028s hns-8myc tagged cells were isolated by immunomagnetic separtion (IMS) from co-crosslinked infected J774A.1 samples for the intracellular ChIP-mini with a biological replicate. [Extracellular ChIP-mini] Mouse macrophage-like cells (J774A.1, RAW 264.7) and human epithelial cells (HeLa, INT407) extracellular S. Typhimurium 14028s WT and hns-8myc tagged cells in DMEM media were crosslinked for the extracellular  ChIP-mini with a biological replicate. [ChIP-exo] E. coli K-12 MG1655 WT and rpoH-8myc tagged cells were grown to mid-log phage, cultured in M9 minimal 0.2% glucose with a biological replicate. For ChIP-exo of mid-log cells, 50 ml of culture media was used to make crosslinking cells (number of cells: 2.4x1010). S. Typhimurium 14028s WT and hns-8myc tagged cells were grown for overnight, cultured in LB media with a biological replicate. For ChIP-exo of overnight-grown cells, about 2.4X1010 cells was used to make crosslinking cells (media volume: 5ml, OD600 : ~6). [ChIP-mini] E. coli K-12 MG1655 WT and rpoH-8myc tagged cells were grown to mid-log phage, cultured in M9 minimal 0.2% glucose with a biological replicate. For ChIP-exo, 6.25 ml (3.0x109), 1.6 ml (7.68x108), 200 ?l (9.6x107), 25 ?l (1.2x107), 10 ?l (4.8x106), and 5 ?l (2.4x106) of culture media was used to make crosslinking cells.