Edta free protease inhibitor

For isolation of ribosomes, HeLa or HEK293 cells were cultured to 80% confluency and scraped off in ice-cold PBS. Cells were lysed in polysome lysis buffer (20 mM Tris-HCl, 150 mM KCl, 5 mM MgCl₂, 0.5% NP40, 2 mM DTT, Roche EDTA-free Protease Inhibitor, and murine RNase inhibitor (New England Biolabs, #M0314)) and incubated while rotating for 10 min at 4 °C. Lysates were cleared by centrifugation at 14,000 g for 10 min and supernatants were recovered and supplemented with 25 µM Hemin (Sigma-Aldrich, #51280). Cleared lysates were loaded on top of a 500 µl 34% sucrose cushion in hypotonic buffer (10 mM HEPES, 10 mM KAc, 0.5 mM MgAc₂) and centrifuged for 240,000 g, 2 h 15 min, 4 °C. Pellets containing ribosomes were washed thoroughly and resuspended in 0.2 X of loaded lysate volume in hypotonic ribosome buffer (20 mM HEPES, 10 mM NaCl, 25 mM KAc, 1.1 mM MgAc₂).

Brains of three- to four-month-old mice were dissected in TBS buffer with 100 μg/ml cycloheximide and immediately frozen in dry ice. Caudate putamen tissues from three mice of mixed gender (1:2 or 2:1 male:female ratio) were pooled; 2.5% of total lysate was subjected to Western blotting to ensure sufficient expression of transgene. The collected samples were homogenized in lysis buffer [10 mm Tris pH 7.5, 150 mm NaCl, 5 mm MgCl₂, 0.5 mm DTT, 100 μg/ml cycloheximide, EDTA-free protease inhibitor (Roche), and 40 U/ml murine RNase Inhibitor (NEB)] with 12 strokes of high-speed motorized homogenizer (Glas-Col GT series) at 40% power. The lysates were briefly centrifuged for 10 min at 2000 × g. The supernatant was transferred to a new tube, added NP-40-1% final concentration, incubated 5 min on ice. The samples were centrifuged again for 10 min at 20,000 × g. The lysates were incubated in ice for 15 min, and centrifuged for 10 min at 20,000 × g. Total RNA concentration of lysate was measured by Qubit RNA BR Assay (Life Technologies), and the same amount of RNA was used across samples. The supernatant was split into two tubes for ribosome footprinting and RNA-seq library generation.