seed cells (SHSY-5Y) in 10 cm dishes at a density of 6 x 10^6 cells per plate.

incubate for 24h 0m 0s

wash cells 2 times with 1X PBS

harvest cells with 1x PBS + PI cocktail (PBS + PI buffer)

1000x g and resuspend in400µL + PI buffer

divide 400µL resuspended cells over 2 vials

tube 1: add 1 µM disuccinimidyl glutarate (DSG; Thermo Fisher Scientific)

tube 2: add 1μM dithiobis succinimidyl propionate (DSP; Thermo Fisher Scientific

incubate 0h 30m 0s at 37°C

Preparation of 50X DSP, 50X DSG

50X DSP: 2.02mg into 100µL DMSO

50X DSG: 1.632mg into 100µL DMSO

Note: Prepare the crosslinkers freshly before use

quenche reaction for 0h 15m 0s on 37Room temperature in both tubes with 20millimolar (mM) Tris (7.4 )

sonicate cells 2 times at 30 Hz with 15 ON-OF intervals of 0h 0m 10s (1s pulses)