GSM5354102: 3C-seq of WT cells after 24 h growth in YEME liquid medium at 30°C in the presence of congocidine 1µg/ml; Streptomyces ambofaciens ATCC 23877; OTHER
Spores of S. ambofaciens ATCC 23877 (≈ 4.107) were inoculated in 200 ml of MP5 or YEME liquid medium. In these media, bacterial growth was monitored by opacimetry. At the indicated time point, cells were harvested from 100 ml samples of culture, adjusted to an OD600 nm of about 0.15 (with the appropriate fresh medium) and fixed by adding formaldehyde solution (3 % final concentration). Cells were then incubated under moderate agitation for 30 min at room temperature and 30 min more at 4°C. Glycine (250 mM final concentration) was added, and the bacteria incubated 30 min at 4°C with moderate agitation. Cells were then harvested by centrifugation for 10 min at 4,000 g at 4°C. The cells were gently suspended in 50 ml of PBS 1X and then again harvested by centrifugation for 10 min at 4,000 g at 4°C. This washing step was repeated once before suspending the cells in 1 ml of PBS before final harvesting by 10 min at 4,000 g at 4°C. The dry pellets were stored at -80°C until use.   Frozen pellets of exponentially grown cells were thawed, suspended in 600 µl Tris 10 mM EDTA 0.5 mM (TE) (pH 8) with 4 µl of lysozyme (35 U/µl; Tebu Bio) and incubated at RT for 20 min. For the samples collected after 48h, the pellets were thawed, suspended in TE (pH 8) with 4 µl of lysozyme (35 U/µl; Tebu Bio) for 45 min and then homogenized with a Bioruptor sonication device (3 cycles of 30 seconds, with a pause of 30 second pause for each). Then, for both 24 h and 48 h samples, SDS was added to the mix (final concentration 0.5%) of cells and incubated for 10 minutes at RT. 500 µl of lysed cells were transferred to a tube containing 4.5 ml of digestion mix (1X NEB 3 buffer, 1% triton X-100) and 100 µl of the lysed cells were transferred to a tube containing 0.9 ml of digestion mix. 800 units of SalI were added to the 5 ml digestion mix. Both tubes were then incubated for 2 h at 37°C and 250 units of SalI were added to the 5 ml tube and further incubated, 2 h at 37°C. To stop the digestion reaction, 4 ml of the digestion mix were immediately centrifuged for 20 min at 20,000 g, and pellets suspended in 4 ml of sterile water. The digested DNA (4 ml in total) was split into 4 aliquots and diluted in 8 ml ligation buffer (1X ligation buffer NEB 3 (without ATP), 1 mM ATP, 0.1 mg/ml BSA, 125 Units of T4 DNA ligase 5 U/µl). Ligation was performed at 16°C for 4 h, followed by incubation overnight at 65°C with 100 µl of proteinase K (20 mg/ml) and 100µl EDTA 500 mM. DNA was then precipitated with an equal volume of 3 M Na-Acetate (pH 5.2) and two volumes of iso-propanol. After one hour at -80°C, DNA was pelleted and suspended in 500 µl 1X TE buffer. The remaining 1ml digestion mix with or without SalI were directly incubated with 100 µl of proteinase K (20 mg/ml) overnight at 65°C. Finally, all the tubes were transferred into 2 ml centrifuge tubes (8 tubes), extracted once with 400 µl phenol-chloroform pH 8.0, precipitated, washed with 1 ml cold ethanol 70% and diluted in 30 µl 1X TE buffer in the presence of RNAse A (1 g/ml). Tubes containing the ligated DNA (3C libraries) were pooled. The efficiency of the 3C preparation was assayed by running aliquots of the 3C-librairies, the digested DNA or the non-digested DNA on 1% agarose gel. Finally the 3C libraries were quantified on the gel using QuantityOne software (BioRad). Approximately 5 µg of a 3C library was suspended in water (final volume 130 µL) and sheared using a Covaris S220 instrument (Duty cycle 5, Intensity 5, cycles/burst 200, time 60 sec for 4 cycles). The DNA was purified using Qiaquick® PCR purification kit, DNA ends were prepared for adapter ligation following standard protocols (see Cournac 2016).  Custom-made adapters (Marbouty 2015) were ligated overnight at 4°C. Ligase was inactivated by incubating the tubes at 65°C for 20 minutes. To purify DNA fragments ranging in size from 400 to 900 pb, a PippinPrep apparatus (SAGE Science) was used. For each library, one PCR reaction of 12 cycles was performed (using 2 µL of 3C library, 0.2 µM Illumina primers PE1.0 and PE2.0 and xx units of Taq Phusion [Finnzymes]). The PCR product was purified on Qiagen MinElute columns and primers dimers were removed from the 3C library by using AMPure XP beads following manufacturer's protocol (Beckman Coulter). Finally, libraries were subject to paired-end sequencing on an Illumina sequencer (NextSeq).