GSM8128527: EhoTldR_input; Escherichia coli; RIP-Seq
E. coli cells were scraped and spread onto the wall of a 50 ml conical tube in a thin layer, to allow for efficient resuspension and crosslinking in the next step.Crosslinking and immunoprecipitation generally followed our previous ChIP-seq protocols (Hoffmann, Kim, Beh et al., 2022; Meers et al., 2023), based on previously established protocols (Bonocora and Wade, 2015; Davis et al. 2011). 1 ml of 37% formaldehyde (Fisher Scientific) was added to a separate tube containing 40 ml of LB medium (~1% final concentration), and the solution was mixed immediately by inverting. This solution was added to the conical tube containing the scraped cells, and the cells were fully resuspended in the LB-formaldehyde solution by vigorous vortexing at room temperature. Crosslinking was performed by gently shaking at room temperature for 20 min. To stop crosslinking, 4.6 ml of 2.5 M glycine (~0.25 M final concentration) were added, followed by 10 min incubation with gentle shaking. Cells were pelleted at 4 °C by spinning at 4,000 x g for 8 min. The following steps were performed on ice, using buffers that had been sterile-filtered through a 0.22 µm filter. The supernatant was discarded and pellets were fully resuspended in 40 ml TBS buffer (20 mM Tris-HCl pH 7.5, 0.15 M NaCl) by vortexing. After another spin at 4,000 x g for 8 min at 4°C, the supernatant was removed and the pellet again resuspended in 40 ml TBS buffer. Next, the OD at 600 nm was measured for a 1:1 mixture of the cell suspension and fresh TBS buffer, and a standardised volume equivalent to 40 ml of OD600 = 0.6 was aliquoted into new 50 ml conical tubes. A final 8 min spin at 4,000 x g and 4 °C was performed, cells were pelleted, and the supernatant was discarded. Residual liquid was removed by briefly inverting the tube, and cell pellets were flash frozen using liquid nitrogen and stored at -80 °C or kept on ice for the subsequent steps. Bovine serum albumin (GoldBio) was dissolved in 1X PBS buffer (Gibco) and sterile-filtered to generate a 5 mg/ml BSA solution. For each sample, 25 µl of Dynabeads Protein G (Thermo Fisher) slurry (hereafter referred to as 'beads' or 'magnetic beads') were prepared for immunoprecipitation. Beads from up to 250 µl of the initial slurry were processed together in a single tube, and washes were performed at room temperature, as follows: The slurry was transferred to a 1.5 ml tube and placed onto a magnetic rack until the beads had fully settled. The supernatant was removed carefully, 1 ml BSA solution was added, and beads were fully resuspended by vortexing, followed by rotating for 30 seconds. This was repeated for three more washes. Finally, beads were resuspended in 25 µl (× n samples) of BSA solution, followed by addition of 4 µl (× n samples) of monoclonal ANTI-FLAG M2 antibody produced in mouse (Sigma-Aldrich). The suspension was moved to 4 °C and rotated for >3 h to conjugate antibodies to magnetic beads. While conjugation was proceeding, crosslinked cell pellets were thawed on ice, resuspended in FA Lysis Buffer 150 (50 mM HEPES-KOH pH 7.5, 0.1% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 1 mM EDTA, 1% (v/v) Triton X-100, 150 mM NaCl) with protease inhibitor cocktail (Sigma-Aldrich) and transferred to a 1 ml milliTUBE AFA Fiber (Covaris). Samples were sonicated on a M220 Focused-ultrasonicator (Covaris) with the following SonoLab 7.2 settings: min. temp. 4°C, set point 6 °C, max. temp. 8°C, peak power 75.0, duty factor 10, cycles/bursts 200, 17.5 min sonication time. After sonication, samples were cleared of cell debris by centrifugation at 20,000 x g and 4 °C for 20 min. The pellet was discarded, and the supernatant (~1 ml) was transferred into a fresh tube and kept on ice for immunoprecipitation. For non-inmunoprecipitated input control samples, 10 µl (~1%) of the sheared cleared lysate were transferred into a separate 1.5 ml tube, flash-frozen in liquid nitrogen, and stored at -80 °C. After >3 h, the conjugation mixture of magnetic beads and antibody was washed four times as described above, but at 4 °C. Next, the beads were resuspended in 30 µl (× n samples) FA Lysis Buffer 150 with protease inhibitor, and 31 µl of resuspended antibody-conjugated beads were mixed with each sample of sheared cell lysate. Samples were rotated overnight for 12–16 h at 4 °C for immunoprecipitation of FLAG-tagged proteins. The next day, tubes containing beads were placed on a magnetic rack and the supernatant was discarded. Then, six bead washes were performed at room temperature as follows, using 1 ml of each buffer followed by sample rotation for 1.5 min: (1) Two washes with FA Lysis Buffer 150 (without protease inhibitor); (2) one wash with FA Lysis Buffer 500 (50 mM HEPES-KOH pH 7.5, 0.1% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 1 mM EDTA, 1% (v/v) Triton X-100, 500 mM NaCl); (3) one wash with ChIP Wash Buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 1 mM EDTA, 1% (v/v) Triton X-100, 500 mM NaCl); and (4) two washes with TE Buffer 10/1 (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were then placed on a magnetic rack, the supernatant was removed, and beads were resuspended in 200 µl of ChIP Elution Buffer (1% (w/v) SDS, 0.1 M NaHCO3), freshly made on the day of the washes. To release protein-DNA complexes from beads, the suspensions were incubated at 65 °C for 1.25 h with gentle vortexing every 15 min to resuspend settled beads. During this incubation, the non-immunoprecipitated input samples were thawed, and 190 µl of ChIP Elution Buffer was added, followed by the addition of 10 µl of 5 M NaCl. After the 1.25 h incubation of the immunoprecipitated samples was complete, tubes were placed back onto a magnetic rack, and the supernatant containing eluted protein-DNA complexes was transferred to a new tube. 9.75 µl of 5 M NaCl were added to ~195 µl of eluate, and samples (both immunoprecipitated and non-immunoprecipitated controls) were incubated at 65°C overnight (without shaking) to reverse-crosslink proteins and DNA. The next day, samples were mixed with 1 µl of 10 mg/ml RNase A (Thermo Fisher) and incubated for 1 h at 37 °C, followed by addition of 2.8 µl of 20 mg/ml Proteinase K (Fisher Scientific) and 1 h incubation at 55 °C. After the addition of 1 ml of buffer PB (QIAGEN recipe), samples were purified using QIAquick spin columns (QIAGEN) and eluted in 40 ul TE Buffer 10/0.1 (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA). RIP-seq: Cells harvested for RIP-seq were cultured as described for ChIP-seq using an E. coli K12 MG1655 strain expressing sfGFP and mRFP (sSL3580). Colonies from a single plate were scraped and resuspended in 1 ml of TBS buffer (20 mM Tris-HCl pH 7.5, 0.15 M NaCl). Next, the OD600 was measured for a 1:20 mixture of the cell sus- pension and TBS buffer, and a standardized amount of cell material equivalent to 20 ml of OD600 = 0.5 was aliquoted. Cells were pelleted by centrifugation at 4,000 g and 4 °C for 5 min. The supernatant was discarded, and pellets were stored at -80 °C. Antibodies for immunoprecipitation were conjugated to magnetic beads as follows: for each sample, 60 μl Dynabeads Protein G (Thermo Fisher Scientific) were washed 3× in 1 ml RIP lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM KCl, 1 mM MgCl2, 0.2% Triton X-100), resuspended in 1 ml RIP lysis buffer, and combined with 20 μl anti-FLAG M2 antibody (Sigma-Aldrich), and rotated for >3 h at 4 °C. Antibody-bead complexes were washed 3× to remove unconjugated antibodies, and resuspended in 60 μl RIP lysis buffer per sample. Flash-frozen cell pellets were resuspended in 1.2 ml RIP lysis buffer supplemented with cOm- plete Protease Inhibitor Cocktail (Roche) and SUPERase•In RNase Inhibitor (Thermo Fisher Scientific). Cells were then sonicated for 1.5 min total (2 sec ON, 5 sec OFF) at 20% amplitude. Lysates were cen- trifuged for 15 min at 4 °C at 21,000 g to pellet cell debris and insoluble material, and the supernatant was transferred to a new tube. At this point, a small volume of each sample (24 μl, or 2%) was set aside as the “input” starting material and stored at -80 °C. For immunoprecipitation, each sample was combined with 60 μl antibody-bead complex and rotated overnight at 4 °C. Next, each sample was washed 3× with ice-cold RIP wash buffer (20 mM Tris- HCl, 150 mM KCl, 1 mM MgCl2). After the last wash, beads were resuspended in 1 ml TRIzol (Thermo Fisher Scientific) and RNA was eluted from the beads by incubating at RT for 5 min. A magnetic rack was used to separate beads from the supernatant, which was transferred to a new tube and combined with 200 μl chloroform. Each sample was mixed vigorously by inversion, incubated at RT for 3 min, and centrifuged for 15 min at 4 °C at 12,000 g. RNA was isolated from the upper aqueous phase using the RNA Clean & Concentrator-5 kit (Zymo Research). RNA from input samples was isolated in the same manner using TRIzol and column purification. High-throughput sequencing library preparation was per- formed as described below for total RNA-seq of Enterobacter strains. Libraries were sequenced on an Illumina NextSeq 550 in paired-end mode with 75 cycles per end. Total RNA-seq: Enterobacter cloacae strains (sSL3710, sSL3711, and sSL3712) were obtained from a CDC isolate panel (Enterobacterales Carbapenemase Diversity; CRE in ARIsolateBank), and an Enterobacter sp. BIDMC93 (sSL3690) was kindly provided by Ashlee M. Earl at the Broad Institute; strain information is listed in Supplementary Table 1. Biological replicates were obtained by isolating 3 individual clones of each Enterobacter strain on LB-agar plates and using these to inoculate overnight cultures in liquid LB media. All strains were grown at 37 °C without antibiotics and with agitation when in liquid medium (240 rpm), in a BSL-2 environment. For total RNA-seq library preparation, RNA was purified from 2 mL of exponentially growing cultures of sSL3690, sSL3710, sSL3711, and sSL3712, since RT-qPCR analyses of fliC expression showed that the TldR-mediated was more robust in exponential than in stationary phase. RNA was extracted using TRIzol and column purification (NEB Monarch RNA cleanup kit), and samples were then individually diluted in NEBuffer 2 (NEB) and fragmented by incubating at 92 °C for 1.5 min. The fragmented RNA was simultaneously treated with RppH (NEB) and TURBO DNase (Thermo Fisher Scientific) in the presence of SUPERase•In RNase Inhibitor (Thermo Fisher Scientific), in order to remove DNA and 5′ pyrophosphate. For further end repair to enable downstream adapter ligation, the RNA was treated with T4 PNK (NEB) in 1× T4 DNA ligase buffer (NEB). Samples were column-purified using RNA Clean & Concentrator-5 (Zymo Research), and the concentration was determined using the DeNovix RNA As- say (DeNovix). Illumina adapter ligation and cDNA synthesis were performed using the NEBNext Small RNA Library Prep kit, using 100 ng of RNA per sample. High-throughput sequencing was performed on an Illumina NextSeq 550 in paired-end mode with 75 cycles per end.