CLIP-seq of CsrA foci in Enteropathogenic E. coli
CLIP-seq of CsrA foci in Enteropathogenic E. coli
RNA was extracted from enriched CsrA foci (CsrA-FLAGx3-GFP, strain LA9743) and from wild-type EPEC, as a negative control for non-specific bead-associated RNA.  Cultures of EPEC expressing CsrA-GFP, or CsrA-FLAG-GFP, or as negative control, wild-type EPEC, were grown in DMEM standing conditions at 37°C to D600 0.6, pelleted, and gently lysed on ice using in a lysis buffer (200 mM Tris pH 8, 20% sucrose, 2% protease inhibitor without EDTA (Roche Cat. 1-836-170), recombinant RNase Inhibitor 0.1 U/µl (Cat. 2313A Takara), DNase I 25 µg/ml, 1% IGEPAL (Cat. I8896 Sigma) and lysozyme 0.25 mg/ml). The lysate was cleared from the cell debris by centrifugation (4°C, 18,400 g, 10 min) and the foci-containing supernatant was collected.  The foci were then pelleted by ultracentrifugation (4°C, 186,000 g for 1 hour, TLA-55 Beckman Coulter Optima TLX Ultracentrifuge). The pellets were gently suspended with washing buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, EDTA free protease inhibitor and recombinant RNase inhibitor 0.1 U/ul) and pelleted again as before.  The pellets containing the CsrA-GFP foci were then gently suspended in 500 µl of washing buffer and subjected to affinity purification. In case of CsrA-GFP purification the foci were immuno-precipitated with anti-GFP trap particles (gtd-20, Chromotek). To purify the CsrA-FLAG-GFP the foci were first immuno-precipitated with anti-FLAG beads (Cat M8823, Sigma), eluted with FLAG peptide (Sigma Cat F4799), and the eluate was subjected to second precipitation step using anti-GFP trap particles (gtd-20, Chromotek) The RNA from three biological replicates for each strain was extracted using TRI reagent (T9424, Sigma) according to the manufacturer's instructions.  RNAtag-Seq was used (Shishkin et al, Nature Methods, 2015) with several modifications as described in Melamed et al., Nature protocols, 2018, starting from step #53.