The spleens were aseptically removed from mice (n = 5/group) and disrupted by passage through a sterile plastic strainer.
P. putida cell pellets were disrupted by passage through French press.
Any remaining intact tissue was disrupted by passage through a 21G needle.
Cells were disrupted by passage through a cooled French pressure cell with a pressure difference of 139 MPa.
Samples were homogenized and further disrupted by passage through a 21-gauge needle (8 to 10 times).
Cell pellets were resuspended in 2 ml cell culture medium and disrupted by passage several times through a hypodermic needle (25G × 1 in, 0.5 mm × 25 mm; Terumo, Somerset, NJ, USA).
For mechanical fractionation, infected HeLa cells were disrupted by passage through a 22G needle in lysis buffer containing 10 mM HEPES (pH 7.4), 250 mM sucrose, 0.5 mM EDTA, and Complete EDTA-free protease inhibitor cocktail (Roche).
P. aeruginosa cells were resuspended in 50 mM Tris/HCl, pH 8.0, containing 15 mM EDTA and disrupted by passage through a French-Pressure cell at 1000 psi (1 psi = 6.9 kPa).
Bacteria (from 1 l of culture) were diluted in 20 mM Tris/HCl pH 7.6, containing 5% sucrose (w/v), 1 mM dithiothreitol (DTT), 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and one tablet Complete Protease inhibitor cocktail, EDTA-free (Roche)/25 ml, and disrupted by passage through a French press (type FA-073; SLM Instruments, Urbana, IL) at 69 MPa.
The cell pellet was then lysed in the same volume of Lysis buffer on ice for 10 min. The nuclear membrane was disrupted by passage through a 28 G needle and then centrifuged at 14,000 g for 15 min. Supernatants were collected as total cell extracts.
Considering the stability of heterochromatin and its restrictive role in the reprogramming process, it is important to consider that cells undergo dynamic cell cycle-associated chromatin changes with the existing chromatin structure disrupted by passage of the replication fork during S phase (MacAlpine & Almouzni, 2013).
