GSM6735218: PB019.TCAACAGG; Mus musculus; RNA-Seq
Nuclei were prepared from the ventral midbrain of mice at 48 hours after injection of saline or IFN-γ. Mice were sacrificed by cervical dislocation and brains were rapidly extracted and submerged in ice-cold phosphate-buffered saline (PBS). Brains were placed on an ice-cold brain matrix (Zivic Instruments) and separated into 0.5-1.0 mm sections using ice cold razor blades. Ventral midbrain tissue was dissected from slices between approximately -2.5 mm to -3.75 mm AP to Bregma. First, the cortex, hippocampi, and any hypothalamus or white matter ventral to the midbrain were removed. A single horizontal cut was made just dorsal to the rostral linear nucleus and all dorsal tissue was discarded. The remaining tissue containing the SN/VTA was flash frozen on liquid nitrogen and stored at -80°C.  Nuclei preparation and sorting were conducted as described by Krishnaswami et al. (2016) with only minor modifications. Frozen tissue was thawed in nuclear isolation medium 1 (NIM1: 250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris, pH 8.0) supplemented with 1 mM DTT, 0.2 U/µL SUPERaseIN, 1x EDTA-free Protease Inhibitor Cocktail (Roche), and 0.1% Triton X-100. Tissue was homogenized on ice in a glass-glass dounce homogenizer with 30 gentle strokes of loose and tight clearance pestles. All subsequent purification steps were performed on ice or at 4oC unless otherwise specified. Lysates were spun at 500xg for 5 minutes, resuspended in NIM1 supplemented as above and also with 1% BSA, and passed through a 40µm cell strainer cap. Nuclei were centrifuged again at 500xg for 5 minutes and resuspended in staining buffer (PBS supplemented with 1% BSA, 5 mM MgCl2, 0.2 U/µL SUPERaseIN, 1 µg/mL propidium iodide, and 2 µg/mL mouse anti-NeuN AlexaFluor488). After 30 minutes incubation on ice, nuclei were centrifuged at 500xg for 5 minutes and resuspended in 0.5 mL of FACS buffer (PBS + 0.04% BSA, 0.2 U/µL SUPERaseIN, and 1 µg/mL propidium iodide) for sorting.    Nuclei sorting was conducted on a BD Influx (BD Biosciences) using a 100 μm nozzle at 11.1 psi. Single nuclei were gated first using FSC-H vs. FSC-A and then on propidium iodide fluorescence. NeuN/AlexaFluor488 fluorescence was used to establish NeuN- and NeuN+ gates on the single nuclei population. Nuclei were sorted directly into 96 well plates (for bulk nuclei RNA-seq with 96-well plate, pooled library construction) or into 1.5 mL Eppendorf tubes that had been previously coated with 0.1% BSA in PBS overnight at 37oC (for snRNA-seq). Low-input bulk RNA-seq: The protocol for plate based, 3' end unique molecular indicator (UMI)-based RNA sequencing of single cells has been described previously (Snyder et al., 2019) as well as modifications to accommodate ultra-low input samples (Hobson et al., 2022b). See Supplementary File 1 for sequences of all custom primers and oligonucleotides used in this protocol. For neuronal and glial nuclei analysis in Figure 1-2, 100 NeuN- or NeuN+ nuclei were sorted directly into the wells of a 96 well plate containing 3 µL of nuclease-free water containing 1 U/µL SUPERaseIN (sort volume ~3 µL for total volume of ~6 µL). After adding 1.5 µL of 10 µM barcoded RT primer (Integrated DNA Technologies), primer annealing was performed at 72°C for 3 minutes. Reverse transcription was performed by adding 7.5 µ