title: GSM5551342: Cas8_crRNA-291_ChIP-seq_paired_raw; Escherichia coli; ChIP-Seq
library_construction_protocol: IPTG-induced 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 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 OD600 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 either single- or paired-end mode on the Illumina MiniSeq and NextSeq platform. For each ChIP-seq sample, >1,800,000 raw reads (including genomic and plasmid-mapping reads) were obtained.