Nuclei Isolation

Prepare buffers and filter sterilize, add RNAse inhibitor fresh NP40 Lysis Buffer (NST): 0.1% NP-40 Alternative (or NP-40), 10millimolar (mM) Tris, 146millimolar (mM) NaCl, 1millimolar (mM)CaCl2, 21mM MgCl2, 40U/mL of Protector RNAse inhibitor (add fresh day of) ST Wash Buffer: (10mM Tris, 146Mass Percent NaCl, 1millimolar (mM) CaCl2, 21mM MgCl2), 0.01% BSA, 40U/mL of Protector RNAse inhibitor (add fresh day of) ST Staining buffer (ST-SB): 2%BSA, 0.02%Tween-20, 10millimolar (mM) Tris, 146millimolar (mM)NaCl, 1mM CaCl2, 21mM MgCl2), 40U/mL of Protector RNAse inhibitor (add fresh day of) Note: Keep tissues/homogenate and buffers on ice throughout the protocol. Pre-cool the centrifuge to 4°Cand keep at 4°C for all steps.

Tissue collection a) Sacrifice and rapidly decapitate mice. Using chilled brain matrix (Ted Pella), cut a thick coronal section from 5mm back from start of cortex until start of cerebellum. Store section in ice-cold Hibernate-A media in 10cm plate on ice until all brains have been dissected. b) Use a razor blade to dissect out midbrain and collect tissue, placing directly into dounce homogenizer.

Tissue lysis and homogenizing a) For each sample to barcode and pool: prepare a separate homogenizer and douncing pestles (loose and tight). Add 1mL NST buffer to the tube with tissue and transfer to dounce homogenizer and keep on ice. b) with a total volume of 1mL, dounce 20 times with the loose pestle followed by 20 times with the tight pestle. c) Add 1mL of ST wash buffer, filter through 30µm filters (Milentyi Biotec 130-041-407) and transfer filtered homogenate to a 15mL tube. d) Rinse the homogenizer with 3x 1mLof ST wash buffer, filter through 30µm filters and add to the filtered homogenate to add up to a final volume of 5mL. e) Immediately spin down at 500g for 0h 5m 0s at 4°C to pellet the nuclei in swing bucket rotor f) Remove supernatant g) Resuspend nuclei in 200µl of ST-SB and transfer to lo-bind 1.5mLtube and wash out the original tube with an additional 1mL and transfer to the same 1.5ml tube. h) Wash by spinning down for 0h 5m 0s at 500g at 4°Cand resuspending in 1.2mL ST-SB twice, for a total of 3 washes i) resuspend in 1mL ST-SB

Proceed with sorting nuclei for GFP+ nuclei via MACS Tyto