Tissues were homogenized in NP40 lysis buffer and the lysates were spun at 13 000 rpm for 15 min and the protein concentration of supernatant was determined using a BCA Protein Assay kit (Thermo Scientific, 23 237).
Retinal homogenates were transferred to 15-ml centrifuge tubes and sequentially overlaid with 3.0 ml of 42%, 3.0 ml of 37%, and 4.0 ml of 32% sucrose dissolved in buffer A. The gradients were spun at 82,000 × g for 1 h at 4°C.
To improve recovery from the columns, water or elution buffer was spun into the matrix at 50 g and left to stand for 4 minutes before a 16,000- g centrifugation.
After decapitating by vortexing, equal numbers of frozen heads from each genotype were homogenized and solubilized in ice-cold RIPA buffer for 30 min. Homogenates were spun at 500× g and then 14,000× g for 15 min at 4°C to remove debris [35].
750 uL of buffer PE was added to the column, samples were spun at 13,000 rpm for 60 seconds and flow-through was discarded.
The nuclei were spun at 1000 g and resuspended in storage buffer (50 mmol/l Tris pH8.5, 0.1 mmol/l EDTA, 5 mmol/l MgCl2, 40% glycerol).
After sorting, cells were spun at 3,000 g for 10 min and any FACS buffer was removed.
At the end of the incubation, the agarose beads were spun at 3000×g for 1 min and washed three times with lysis buffer.
In total, 100 µL of beads slurry were spun at 160 g for 5 min and washed once with GUV buffer 0.25% BSA.
Cells were spun at 4°C for 20 min to pellet, and resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH = 8).
