GSM7729916: 3C-seq of D. radiodurans wt cells -Replicate 2; Deinococcus radiodurans; OTHER
To fix DNA-DNA contacts, 1-2 x 109 cell culture were collected by centrifugation and mixed with fresh formaldehyde for 30 minutes at 25C (7% final concentration) followed by 30 minutes at 4°C. Formaldehyde was then quenched with a final concentration of 0.25 M glycine for 20 minutes at 4°C. Fixed cells were collected by centrifugation and washed once with double distilled water. The cell pellets by liquid nitrogen flash freezer were stored at −80°C until use. Frozen pellets were thawed in the ice box, resuspended and lysed by incubation with lysozyme at 37°C for 30 min. The reaction was quenched by adding 10% SDS and incubating for 5 min at 65°C. 50 μL of lysed cells were then transferred in one EP tube containing 450 μL of digestion mix (355 μL of double distilled water, 50 μL of 10 × rCutSmart™ Buffer, 25 μL of 25% Triton X-100, and 20 μL of 10 U/μL HpaII enzyme). DNA was digested for 10 hours at 37°C with shaking at 200 rpm/min. For ligation, the reaction was mixed with 7945 μL of double distilled water, 1000 μL of 10 × T4 DNA Ligase Reaction Buffer (NEB), 50 μL of 20 mg/mL BSA (TaKaRa), 500 μL of 20% Triton X-100, and 5 μL of 400 U/μl T4 DNA ligase (NEB). The ligation reaction was incubated for 4 h at 16°C with occasional inversion of the tube (every 1 h). After the ligation was completed, the reaction was mixed with 100 μL of 0.5 M EDTA and 50 μL of 20 mg/ml proteinase K (TransGen) and incubated at 58°C for 4 h. Precipitation of DNA was performed by standing overnight at 4°C in presence of 3 M Na-Acetate (pH 5.2) and iso-propanol. The following day, the DNA was extracted through using DNA Extraction Reagent (SOLARBIO). The obtained DNA was washed with 500 μL 75% cold ethanol, dissolved in 40 μl 1 × TE buffer containing 0.1 mg/ml RNase A and incubated for 30 min at 37°C. Successful proximity ligation was confirmed by running 5 μL of the DNA on a gel. The resulting DNA was andomly fragmented, followed by end repair and addition of an 'A' base at the 3' end. After ligating the adapters, PCR amplification was performed for each library. The PCR products were then purified, circularized into single-stranded templates, and subjected to paired-end sequencing on the BGI DNBSEQ platform.