GSM8892705: A4441_011625; Escherichia coli; ChIP-Seq
For ChIP-seq, liquid LB overnight E. coli cultures were made up to 40 ml of volume using LB media. 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 the tubes containing 40 ml of overnight culture (~1% final concentration), and the solution was mixed immediately by inverting and vortexing. 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). For ChIP-seq, Illumina libraries were generated for immunoprecipitated and input samples using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB). Sample concentrations for ChIP-seq were determined using the DeNovix dsDNA Ultra High Sensitivity Kit. Starting DNA amounts were standardised such that an approximately equal mass of all input and immunoprecipitated DNA was used for library preparation. After adapter ligation, a single PCR amplification was performed to add Illumina barcodes, and ~450 bp-sized DNA fragments were selected using a two-sided AMPure XP bead (Beckman Coulter) size selection, as follows: The volume of barcoded immunoprecipitated and input DNA was brought up to 50 µl with TE Buffer 10/0.1; in the first size selection step, 0.55X AMPure beads (27.5 µl) were added to the DNA, the sample was placed on a magnetic rack, and the supernatant was discarded and the AMPure beads were kept; in the second size selection step, 0.35X AMPure beads (17.5 µl) were added to the DNA, the sample was placed on a magnetic rack, and the AMPure beads were discarded and the supernatant was kept. The concentration of DNA was determined for pooling using the DeNovix dsDNA High Sensitivity Kit. Illumina libraries were sequenced in paired-end mode on the Illumina NextSeq 500 and Element Biosciences Aviti Cloudbreak Freestyle platform. The extraction protocol was performed as described in Tang et al. 2024. After overnight growth, cells equivalent to 20 ml of OD600=0.5 were aliquoted. Cells were harvested by centrifugation at 4000 × g for 10 min at 4°C, and the supernatant was removed. The pellet was washed with cold TBS (20 mM Tris-HCl, pH 7.5 at 25°C, 150 mM NaCl) and spun down again as before. The supernatant was removed, and the pellet was flash-frozen in liquid nitrogen and 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 IP lysis buffer (20 mM Tris-HCl, pH 7.5 at 25°C, 150 mM KCl, 1 mM MgCl2, 0.2% Triton X-100), resuspended in 1 ml IP lysis buffer, combined with 20 μl anti-FLAG M2 antibody (Sigma-Aldrich, F1804), and rotated for >3 hours at 4°C. Antibody-bead complexes were washed 2× to remove unconjugated antibodies and resuspended in 60 μl of IP lysis buffer per sample. Flash-frozen pellets were thawed on ice and resuspended in 1.2 ml IP lysis buffer supplemented with 1× cOmplete Protease Inhibitor Cocktail (Roche) and 0.1 U μl−1 SUPERase•In RNase Inhibitor (Thermo Fisher Scientific). To lyse cells, samples were sonicated using a 1/8″ sonicator probe for 1.5 min total (2 s ON, 5 s OFF) at 20% amplitude. To clear cell debris and insoluble material, lysates were centrifuged at 21,000 × g for 15 min at 4 °C, and 1 ml supernatant was transferred to a new tube. At this point, two small volumes of each sample (10 μl for RIP-seq and 10 μl for cDIP-seq) were set aside as “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. The next day, each sample was washed 3× with 1 ml ice-cold IP wash buffer (20 mM Tris-HCl, pH 7.5 at 25°C, 150 mM KCl, 1 mM MgCl2), using a magnetic rack to immobilize the beads in between each wash. During the final wash, each sample was separated into two separate 500 μl volumes for downstream RIP or cDIP processing. For RIP elution, the supernatant was removed, and beads were resuspended in 1000 μl TRIzol (Thermo Fisher Scientific). After 5 min incubation at RT, the supernatant containing eluted RNA was transferred to a new tube and combined with 200 μl chloroform. Samples were mixed vigorously by inversion, incubated at RT for 3 min, and centrifuged at 12,000 × g for 15 min at 4°C. RNA was isolated from the upper aqueous phase using the RNA Clean & Concentrator-5 kit (Zymo Research) and eluted in 15 μl RNase-free water. RNA from input samples was isolated in the same manner using TRIzol and column purification. Purified RNA was stored at -80°C before proceeding to library preparation. For RIP-seq library preparation (input and RIP eluates), RNA was fragmented by random hydrolysis by combining 7 μl RNA, 6 μl water, and 2 μl NEBuffer 2, and heating to 92°C for 2 min. To remove DNA and prepare RNA ends for adapter ligation, samples were treated with 2 μl TURBO DNase (Thermo Fisher Scientific) and 2 μl RppH (NEB) in the presence of 1 μl SUPERase•In RNase Inhibitor for 30 min at 37°C. This was followed by treatment with 1 μl T4 PNK (NEB) in 1× T4 DNA ligase buffer (NEB) for 30 min at 37°C. Reactions were column-purified using the Zymo RNA Clean and Concentrator-5 kit and eluted in 10.5 μl RNase-free water. RNA concentrations were quantified using the DeNovix RNA Assay. Illumina sequencing libraries were prepared using the NEBNext Small RNA Library Prep kit. Illumna ibraries were sequenced in paired-end mode on the Illumina NextSeq 500 and Element Biosciences Aviti Cloudbreak Freestyle platform. For RNA-seq, overnight cultures were generally processed as described in Tang et al. 2024. Cells equivalent to 20 ml of OD600 = 0.5 were aliquoted and harvested by centrifugation at 4000 × g for 10 min at 4°C, and the supernatant was removed. The pellet was washed with cold TBS (20 mM Tris-HCl, pH 7.5 at 25°C, 150 mM NaCl) and spun down again as before. The supernatant was removed, and the pellet was flash-frozen in liquid nitrogen and stored at -80°C. Cell pellets were resuspended in 1,000 μl TRIzol (Thermo Fisher Scientific). After 5 min incubation at RT, the supernatant containing eluted RNA was transferred to a new tube and combined with 200 μl chloroform. Samples were mixed vigorously by inversion, incubated at RT for 3 min, and centrifuged at 12,000 × g for 15 min at 4°C. RNA was isolated from the upper aqueous phase using the RNA Clean & Concentrator kit (Zymo Research) and eluted in 15 μl RNase-free water. Purified RNA was stored at -80°C before proceeding to library preparation. For RNA-seq library preparation, RNA was fragmented by random hydrolysis by combining 7 μl RNA, 6 μl water, and 2 μl NEBuffer 2, and heating to 92°C for 2 min. To remove DNA and prepare RNA ends for adapter ligation, samples were treated with 2 μl TURBO DNase (Thermo Fisher Scientific) and 2 μl RppH (NEB) in the presence of 1 μl SUPERase•In RNase Inhibitor for 30 min at 37°C. This was followed by treatment with 1 μl T4 PNK (NEB) in 1× T4 DNA ligase buffer (NEB) for 30 min at 37°C. Reactions were column-purified using the Zymo RNA Clean and Concentrator-5 kit and eluted in 10.5 μl RNase-free water. RNA concentrations were quantified using the DeNovix RNA Assay. Illumina sequencing libraries were prepared using the NEBNext Small RNA Library Prep kit, and libraries were sequenced on an Illumina NextSeq 500 or Element Biosciences Aviti instrument in paired-end mode with 75 or 150 cycles per end.