Model 705 sonic dismembrator

The suspension with the harvested cells was defrosted and 90 µl of DNAse was added (∼2 µg ml⁻¹). The cell suspension was sonicated using a Fisherbrand Model 705 Sonic Dismembrator in three 2 min cycles. The disrupted cells were centrifuged (4°C and 14 000 rev min⁻¹ for 1 h). The protein was then filtered and loaded onto a BioLogic DuoFlow FPLC system (Bio-Rad, USA) equilibrated in buffer A (10 mM Tris, 50 mM sodium formate, 10 mM imidazole pH 8.5) and buffer B (10 mM Tris, 50 mM sodium formate, 500 mM imidazole pH 8.5). The protein was purified by metal-affinity chromatography using a nickel-charged column at a flow rate of 1 ml min⁻¹. The protein was eluted with an increasing gradient of imidazole (0%, 10%, 60% and 100% buffer B; DmmarA was eluted in the 10% and 60% gradients). The purified protein from the 60% imidazole gradient was concentrated to ∼5 ml using Amicon Ultra-15 Centrifugal Filter Units (10 kDa cutoff).
5 ml of protein was loaded onto an ÄKTApure FPLC system (Cytiva, Sweden) equilibrated in gel-filtration buffer (10 mM Tris, 50 mM sodium formate pH 8.5). The protein was purified by size-exclusion chromatography using a Superdex 16/60 200 pg column (GE Healthcare, UK). SDS–PAGE was used to check the purity of fractions from affinity chromatography and size-exclusion chromatography (run at 200 V and 400 mA for 40 min).

The suspension with the harvested cells was defrosted and 90 µl of DNAse was added (∼2 µg ml⁻¹). The cell suspension was sonicated using a Fisherbrand Model 705 Sonic Dismembrator in three 2 min cycles. The disrupted cells were centrifuged (4°C and 14 000 rev min⁻¹ for 1 h). The protein was then filtered and loaded onto a BioLogic DuoFlow FPLC system (Bio-Rad, USA) equilibrated in buffer A (10 mM Tris, 50 mM sodium formate, 10 mM imidazole pH 8.5) and buffer B (10 mM Tris, 50 mM sodium formate, 500 mM imidazole pH 8.5). The protein was purified by metal-affinity chromatography using a nickel-charged column at a flow rate of 1 ml min⁻¹. The protein was eluted with an increasing gradient of imidazole (0%, 10%, 60% and 100% buffer B; DmmarA was eluted in the 10% and 60% gradients). The purified protein from the 60% imidazole gradient was concentrated to ∼5 ml using Amicon Ultra-15 Centrifugal Filter Units (10 kDa cutoff).
5 ml of protein was loaded onto an ÄKTApure FPLC system (Cytiva, Sweden) equilibrated in gel-filtration buffer (10 mM Tris, 50 mM sodium formate pH 8.5). The protein was purified by size-exclusion chromatography using a Superdex 16/60 200 pg column (GE Healthcare, UK). SDS–PAGE was used to check the purity of fractions from affinity chromatography and size-exclusion chromatography (run at 200 V and 400 mA for 40 min).

Cell pellets were resuspended into ice cold Extraction Buffer (25 mM HEPES pH 7.9, 300 mM sucrose, 50 mM NaCl, 1 mM EGTA, 3 mM MgCl₂, 0.5% IGEPAL CA-630) supplemented with cOmplete protease inhibitor cocktail, phosSTOP phosphatase inhibitor cocktail (both from Roche), and 25 mM N-ethylmaleimide (NEM) and tumbled end-over-end for 20 min at 4°C. Samples were then centrifuged at 20 000g for 20 min at 4°C, after which the supernatant was recovered as the soluble fraction. The pellet, representing the chromatin-enriched fraction, was then resuspended into the buffer of choice and sonicated with a Model 705 Sonic Dismembrator fitted with a microtip probe (Fisher Scientific).