GSM4981981: 3C-seq time course t60 of Smc-CC425 and parS at -9kb cells in exponential phase in SMG at 30°C; Bacillus subtilis; OTHER
3C-seq: 3C libraries were prepared as previously described (Marbouty et al., 2015). Minimal media (SMG) was used instead of LB. Briefly, cells were grown in 400 ml of SMG medium to exponential phase (OD = 0.022-0.030) and fixed with fresh formaldehyde (3% final concentration) for 30 minutes at RT, followed by 30 minutes at 4°C and quenched for 30 minutes with 0.25 M glycine at 4°C. Fixed cells were harvested by filtering, washed in fresh SMG, frozen in liquid nitrogen and stored at -80°C until further use. For time-course experiments 2 l preculture was first grown until mid-exponential phase (OD = 0.022-0.030) and next, appropriate culture volumes were added to fresh pre-warmed SMG so that at given time points 2 x 200 ml of culture at mid-exponential could be collected (two technical replicates). The cultures were induced with 2 mM theophylline or 1 mM IPTG, depending on the promoter used, Ptheo or Pspank, respectively. Due to characteristics of the theophylline switch, the pre-culture as well as induction was performed at 30°C. Frozen pellets were resuspended in 600 µl 1x TE and incubated at RT for 20 minutes with 4 µl of Ready-lyze lysozyme (35U/ul, Tebu Bio). Next, SDS was added to a final concentration of 0.5% and incubated at RT for 10 minutes. 50 µl of lysed cells were aliquoted to 8 tubes containing 450 µl of digestion mix (1x NEB 1 buffer, 1% triton X-100, and 100 U HpaII enzyme (NEB)) and incubated at 37°C for 3 hours with constant shaking. Digested DNA was collected by centrifugation, diluted into 4 tubes containing 8 ml of ligation mix (1x ligation buffer: 50 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT), 1 mM ATP, 0.1 mg/ml BSA, 125 U T4 DNA ligase 5 U/ml) and incubated at 16°C for 4 hours. Ligation reaction was followed by O/N decrosslinking at 65°C in the presence of 250 µg/ml proteinase K (Eurobio) and 5 mM EDTA. DNA was precipitated with 1 volume of isopropanol and 0.1 Volume of 3 M sodium acetate (pH 5.2, Sigma) at -80°C for 1 hour. After centrifugation, the DNA pellet was resuspended in 1x TE at 30°C for 20 minutes. Next, DNA was extracted once with 400 µl phenol-chloroform-isoamyl alcohol solution and precipitated with 1.5 volume cold 100% ethanol in the presence of 0.1 volume 3 M sodium acetate at -80°C for 30 minutes. The pellet was collected and resuspended in 30 µl TE with RNaseA at 37°C for 30 minutes. All tubes were pooled and the resulting 3C library was quantified on gel using ImageJ. ChIP-seq: ChIP samples were prepared as described previously (Bürmann et al., 2017). Briefly, cells were cultured in 200 ml of minimal media (SMG) at 37°C until mid-exponential phase (OD=0.022-0.030) and fixed with buffer F (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 0.5 mM EGTA pH 8.0, 1 mM EDTA pH 8.0, 10% (w/v) formaldehyde) for 30 minutes at RT with occasional shaking. Cells were harvested by filtration and washed in PBS. Each sample was adjusted for 2 OD units (2 ml at OD600 = 1) and resuspended in TSEMS lysis buffer (50 mM Tris pH 7.4, 50 mM NaCl, 10 mM EDTA pH 8.0, 0.5 M sucrose and PIC (Sigma), 6 mg/ml lysozyme from chicken egg white (Sigma)). After 30 minutes of incubation at 37°C with vigorous shaking protoplasts were washed again in 2 ml TSEMS, resuspended in 1 ml TSEMS, split into 3 aliquots, pelleted and, after flash freezing, stored at -80°C until further use. For time-course experiments 1 l preculture was first grown until mid-exponential phase (OD = 0.022-0.030) and next, appropriate culture volumes were added to fresh pre-warmed SMG so that at given time points 200 ml of culture at mid-exponential could be processed. The cultures were induced with 2 mM theophylline (Ptheo promoter). Due to characteristics of the theophylline switch, the pre-culture as well as induction was performed at 30°C. For IP of samples for ChIP-seq, each pellet was resuspended in 1 ml of buffer L (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8.0, 1% (v/v) Triton X-100, 0.1% (w/v) Na-deoxycholate, 0.1 mg/ml RNaseA and PIC (Sigma)) and transferred to milliTUBE 1ml AFA Fiber and sonicated in a Covaris E220 water bath sonicator for 5 minutes at 4°C, 100 W, 200 cycles, 10% load and water level 5. Next, lysates were transferred into 2 ml tubes and centrifuged 10 minutes at 21000g at 4°C. 800 µl of supernatant was used for IP and 200 µl was kept as WCE (whole cell extract). For IP, first, antibody serum was incubated with Protein G coupled Dynabeads (Invitrogen) in 1:1 ratio for 2.5 hours at 4°C with rotation. Next, beads were washed in buffer L and 50 µl were aliquoted to each sample tube. Samples were incubated for 2 hours at 4°C with rotation, followed by a series of washes with buffer L, buffer L5 (buffer L containing 500 mM NaCl), buffer W (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate, 1 mM EDTA pH 8.0) and buffer TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0). Finally, the beads were resuspended in 520 µl buffer TES (50 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 1% (w/v) SDS). 300 µl of TES and 20 µl of 10% SDS were also added to WCE. Both tubes were incubated O/N at 65°C with vigorous shaking to reverse formaldehyde crosslinks. Following rabbit polyclonal antibodies (manufactured by Eurogentec) were used: For anti-scpB ChIP time-course experiment COD3 BsScpB-His6 For other anti-scpB ChIP experiments COD33 BsScpB-His6 For anti-parB ChIP experiments COD11 BsParB-His Phenol-chloroform extraction was performed to purify the decrosslinked DNA. Samples were transferred to screw cap 1.5-ml tubes and first mixed vigorously with 500 µl of phenol equilibrated with buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). After centrifugation (10 minutes, RT, 13000 rpm), 450 µl of the aqueous phase was transferred to a new screw cap tube and mixed with equal volume of chloroform, followed by centrifugation. 400ul of aqueous phase was recovered for DNA precipitation with 2.5x volume of 100 % ethanol, 0.1x volume of 3M NaOAc and 1.2 µl of GlycoBlue and incubated for 20 minutes at -20°C. Next, samples were centrifuged for 10 minutes at 20000 g at RT and pellets obtained pellets were resuspended in 10 µl of EB (Qiagen) shaking at 55°C for 10 minutes and finally purified with a PCR purification kit, eluting in 50 uL EB. 3C-seq 1 ug of 3C library was suspended in water (final volume 130 µl) and sonicated using Covaris S220 (following manufacturers recommendations to obtain 500 bp target size). Next, DNA was purified with Qiagen PCR purification kit, eluted in 40 µl EB and quantified using NanoDrop. 1 ug of DNA was processed according to manufacturer instructions (Paired-End DNA sample Prep Kit – Illumina – PE-930-1001), except that DNA was ligated to custom-made adapters for 4 hours at RT, followed by inactivation step at 65°C for 20 minutes. DNA was purified with 0.75x AMPure beads and 3 µl were used for 50 µl PCR reaction (12 cycles). Amplified libraries were purified on Qiagen columns and pair end sequenced on an Illumina platform (HiSeq4000 or NextSeq). ChIP-seq For deep sequencing, the DNA libraries were prepared by Genomic Facility at CIG, UNIL, Lausanne. Briefly, the DNA was fragmented by sonication (Covaris S2) to fragment sizes ranging from 220–250 bp. DNA libraries were prepared using the Ovation Ultralow Library Systems V2 Kit (NuGEN) including 15 cycles of PCR amplification. Five to ten million single end sequence reads were obtained on a HiSeq2500 (Illumina) with 150-bp read length