title: GSM5350259: IP-GY18220-rep1; Deinococcus radiodurans R1; ChIP-Seq
library_construction_protocol: For ChIP seq samples: cells were cross-linked with 1% formaldehyde in TGY2X medium for 25 min at 30°C with continuous shaking. Crosslinking reactions were quenched by the addition of 125 mM glycine for 15 min. Cells were harvested by centrifugation (4000 rpm, 10min, 4°C), washed twice with cold Tris Buffer Solution (TBS) and then resuspended in 3 mL of lysis buffer [160 nM NaCl, 20 mM, Tris-HCl pH7.5, 1 mM EDTA, protease inhibitor cocktail (Roche)]. Cells were disrupted and DNA sheared using a One Shot Cell Disruptor (CellD SARL) to an average size of 100-300 bp (2 rounds of 2.4kbar). Insoluble material was removed by centrifugation at 15,000 rpm for 10 min at 4°C and the supernatant was collected in a sterile microcentrifuge tube. Then, 500 µL of supernatant fluid was added to 25 µL of pre-incubated protein G magnetic beads (ChIP-Adembeads ChIP-Adem-Kit, Ademtech SA) with 5 µg of anti-V5 rabbit polyclonal antibody (ab9116, Abcam) in IP buffer [50 mM HEPES-KOH pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton 100, protease inhibitor cocktail (Roche)]. After overnight incubation at 4°C with rotation, the immunoprecipitates were washed 5 times with washing buffers (ChIP-Adembeads ChIP-Adem-Kit, Ademtech SA).   Immune complexes were eluted in 200 µL of elution buffer. 20 µL The eluted samples (20µL) were saved for control Western blots, and the remainder was incubated for 2 h at 37°C with shaking with 100 µg/mL Proteinase K. Then, the supernatant was incubated overnight at 65°C to reverse cross-linking with 100 µg/mL RNAse A. The DNA was purified using the PCR Clean-up kit (Macherey-Nagel).