Nuclei Isolation and Labeling for Cell-Type Specific Profiling

Nuclei were isolated as described previously^{17 (link)}. For the labeling of glial cell nuclei and cerebellar granule cells, the isolated nuclei were washed once with homogenization buffer (0.25 M sucrose, 150 mM KCl, 5 mM MgCl₂, 20 mM Tricine pH 7.8, 0.15 mM spermine, 0.5 mM spermidine, EDTA-free protease inhibitor cocktail, 1 mM DTT, 20 U ml⁻¹ SUPERase-In RNase inhibitor (ThermoFisher, #AM2696), 40 U ml⁻¹ RNasin ribonuclease inhibitor (Promega, #N2515)). Each washing step constituted of resuspension of nuclei pellet followed by centrifugation (1,000 × g, 4 min, 4 °C). Resuspended nuclei were fixed in Homogenization buffer with 1% formaldehyde for 8 min at room temperature followed by quenching with 0.125 M glycine for 5 min. Following centrifugation, the nuclei were washed once with wash buffer (PBS, 0.05% TritonX-100, 0.5% BSA, 20 U ml⁻¹ Superase-In RNase Inhibitor and 40 U ml⁻¹ RNasin ribonuclease inhibitor) and incubated at room temperature on a shaker in wash buffer for permeabilization and blocking of unspecific binding. Nuclei were washed twice in wash buffer without TritonX-100 and resuspended in 100 µl 40% ethanol containing TrueBlack Lipofuscin Autofluorescence Quencher (Biotium, #23007) for 40–50 seconds. Nuclei were washed twice with wash buffer (w/o TritonX-100) and incubated overnight at 4 °C with the following antibodies: Rb x NeuN-Alexa-647 (1:400, Abcam, #ab190565), Rb x NeuN-Alexa594 (1:400, Abcam, #ab207279), Mm x EAAT1 (1:2,000, Santa Cruz Biotechnology, #sc-515839), Mm x IRF8-PE (1:65, ThermoFisher, #12-9852-82) and Goat x Olig2 (1:300, R&D Systems, #AF2418). After three washes with wash buffer (w/o TritonX-100), the nuclei were incubated for 30–45 min at room temperature with Donkey × Mm-Alexa-488 (1:1,000, ThermoFisher, #A-21202) and Donkey x Goat-Alexa-647 (1:300, ThermoFisher, # A-21447). After three washes with wash buffer (w/o TritonX-100), the nuclei were resuspended in Sorting buffer (PBS, 0.2% BSA, 40 U ml⁻¹ RNasin ribonuclease inhibitor, 0.5 µg ml⁻¹ DAPI) and separated with SONY MA900 Cell Sorter (software ver. 3.0.5). Aggregates of nuclei were excluded based on higher DAPI signal and the following gating strategies were used: neuronal nuclei (647+, 594+, 488−, large), oligodendrocyte nuclei (647+, 594−, 488−, small), microglia nuclei (647−, 594+, 488−, small) and astrocyte nuclei (647−, 594−, 488+, small). A separate sorting experiment was performed for collecting cerebellar granule cell nuclei. For this purpose, nuclei were labeled with Rb x NeuN-Alexa594 (1:400, Abcam, #ab207279) and Mm x ITPR1-Alexa-488 (Santa Cruz Biotechnology, #sc-271197 AF488), and granule cell nuclei were collected (488−, 594+).
For labeling neuronal nuclei, PrimeFlow labeling kit (ThermoFisher, #88-18005-210) was used and fixation and permeabilization were carried out according to manufacturer’s instructions but with 200 U ml⁻¹ Superase-In RNase inhibitor and 400 U ml⁻¹ RNasin ribonuclease inhibitor present at every incubation step. For sorting, the nuclei were resuspended in sorting buffer (PBS, 0.2% BSA, 40 U ml⁻¹ RNasin ribonuclease inhibitor, 0.5 µg ml⁻¹ DAPI). Probes specific to DRD1 (Alexa-647, #VA1-3002351-PF), DRD2 (Alexa-488, #VA4-3083767-PF) and PPP1R1B (Alexa-568, #VA10-3266354-PF) were used to label dMSN (647+, 568+, 488−, large) and iMSN nuclei (647−, 568+, 488+, large). In a separate set of experiments, probes specific to TAC3 (Alexa-647, #VA1-16603-PF), ETV1 (Alexa-488, # VA4-3083818-PF), SST (Alexa-568, # VA10-3252595-PF) and PPP1R1B (Alexa-568, # VA10-3266354-PF) were used to label the nuclei of TAC3+ interneurons (647+, 568−, 488+), PVALB+ interneurons (647−, 568−, 488+), SST+ interneurons (647−, 568+++, 488−) and MSNs (647−, 568+, 488−, large). Probes specific to TRPC3 (Alexa-647, # VA1-3004835-PF), COL6A6 (Alexa-647, #VA1-3014134-PF) and PPP1R1B (Alexa-568, # VA10-3266354-PF) were used in another set of experiments to label cholinergic interneuron nuclei (647+, 568−, large) and MSN nuclei (647−, 568+, large). CA8 probe (Alexa-647, #VA1-3001892-PF) was used for sorting Purkinje neuron nuclei (647+, large). Aggregates of nuclei were always excluded based on higher intensity of DAPI staining. All PrimeFlow target probes were used at a dilution of 1:40.