GSM4560162: 3C-seq of <delta>mks parS+550 cells in exponential phase minimal medium A supplemented with 0.25% citrate; Pseudomonas aeruginosa PAO1; OTHER
3C libraries were generated as described previously (Lioy and Boccard 2018). Briefly, 100 ml of culture was crosslinked with fresh formaldehyde for 30 minutes (5% final concentration) at room temperature (RT) followed by 30 minutes at 4°C. Formaldehyde was quenched with a final concentration of 0.25 M glycine for 20 minutes at 4°C. Fixed cells were collected by centrifugation, frozen on dry ice and stored at -80°C until use. Frozen pellets of ≈ 1-2 x 10e9 cells were defrosted, 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 minutes. SDS was added to the mix (final concentration 0.5%) and the cells incubated for 10 minutes at RT. 50µl of lysed cells were transferred to a tube containing 450µL of digestion mix (1X NEB 1 buffer, 1% triton X-100). This process was repeated 11 times (total 12 tubes with 500 µl). 100 units of HpaII were added to 10 tubes. All the tubes were then incubated for 2 hours at 37°C. To stop the digestion reaction, 8 tubes were immediately centrifuged during 20 min at 20,000 g, and pellets were suspended in 500ul of sterile water. The digested DNA (4 ml in total) was split in 4 aliquots, and diluted in 8 ml ligation buffer (1X ligation buffer NEB 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 hours, followed by incubation overnight (ON) at 65°C with 100 µl of proteinase K (20 mg/ml) and 100ul 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, suspended in 500µl 1X TE buffer. The remaining 4 tubes (2 tubes with HpaII and 2 tubes without restriction enzyme that were also incubated at 37°C with the resto of tubes) were directly incubated with 50 µl of proteinase K (20mg/ml) overnight at 65°C. Finally, all the tubes were transferred into 2 ml centrifuge tubes, extracted twice 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 presence of RNase A (1 mg/ml). Tubes containing the ligated DNA (3C libraries), the digested DNA or the non-digested DNA were pooled into 3 different tubes and the efficiency of the 3C preparation was assayed by running a 1% agarose gel. 3C libraries were quantified on 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).
GEO Accession
GSM4560162