Optiprep density gradient ultracentrifugation

For cell fractionation using 3–30% iodixanol gradient (Optiprep gradient) 1.6 × 10⁷ cells were used as described earlier (Mukherjee et al, 2016). Optiprep (Sigma-Aldrich) solution was used to prepare 3–30% gradient. In brief, the cell pellet was homogenized in a buffer with glass Dounce homogenizer. The lysate was centrifuged at 1,000g twice and the supernatant was loaded on the gradient and centrifuged at 36,000 rpm in a Beckman-Coulter SW60Ti rotor for 5 h. Approximately 400 μl of 10 fractions were collected for RNA and protein.