Mini beadbeater 16 cell disrupter

Slices of tissue weighing 200–350 mg were first homogenized to a final 15% (w/v) concentration in calcium-free and magnesium-free PBS, pH 7.4, by 3 × 75 s cycles with a Mini-Beadbeater 16 Cell Disrupter (Biospec). Homogenates were then diluted to a final 10% (w/v) in 1% (v/v) sarkosyl in PBS, pH 7.4 and re-homogenized. After clarification at 500×g for 5 min, additional sarkosyl was added to a final 2% and the samples were spun at 15,500 rpm at 4 °C for 30 min in Allegra X-22R tabletop centrifuge (Beckman Coulter). To investigate protease-sensitive and resistant fractions of tau, the sample aliquots were digested with Proteinase K (PK) according the rodent and human prion protocol at 1:50 enzyme/total protein (weight/weight) ratio [50 (link), 85 (link), 87 (link)]. The pellet containing sarkosyl-insoluble tau protein was resuspended in PBS, pH 7.4 and divided into two aliquots: the first one was incubated with 100 µg/ml of PK (Amresco) for 1 h at 37 °C with shaking of 600 rpm in an Eppendorf Thermomixer (Eppendorf) and the second one, untreated, was mixed with protease inhibitors cocktail (0.5 mM PMSF and aprotinin and leupeptin at 5 µg/ml, respectively). After blocking PK-treated aliquots with a protease inhibitor cocktail, samples were stored for analysis at − 80 °C.

Coronal sections of human brain tissues were obtained at autopsy and stored at − 80ºC. Slices of frontal cortex (superior and more posterior middle gyri) weighing 200–350 mg were homogenized to a final 15% (w/v) concentration by three 75 s cycles with Mini-beadbeater 16 Cell Disrupter (Biospec) in PBS/2% Sarkosyl, pH 7.4, and clarified at 500xg for 5 min. at 4 °C. Clear supernatant was then transferred to a new tube and stored for future analysis at − 80 °C.

To prepare S. cerevisiae total RNA from cell pellets of 10 ml cultures grown to OD₆₀₀ = 0.5–0.7, glass beads (2–3 x 100 μ l) were added to each cell pellet in 2 ml screw-cap tube, 1 ml TRIzol (Invitrogen) was added and cell sample was vigorously disrupted in Mini Bead Beater 16 Cell Disrupter (Biospec) for 2 min at 25°C. For each sample, 100 μ l of 1-bromo-3-chloropropane (BCP) was added, the sample was vortexed for 15 sec, and incubated at 25°C for 2 min. Each sample was then centrifuged at 16,300 x g for 8 min at 4°C and the upper layer was transferred to a fresh microfuge tube. RNA was precipitated with 500 μ l isopropanol and the sample was vortexed for 10 sec to mix. Total RNA was pelleted by centrifugation at 16,300 x g for 8 min at 4°C. Supernatant was decanted and 1 ml of 75% ethanol was added to wash RNA pellet. Sample was centrifuged at 16,300 x g for 5 min at 4°C. Supernatant was decanted, remaining ethanol was removed, and the RNA pellet was air dried for 15 min. Total RNA was resuspended in 50 μ l diethylpyrocarbonate (DEPC (Sigma))-treated water and stored at-80°C.