GSM6735227: PB019.TACACCAG; 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 µL RT mix to each well (2.81 µL of 40% polyethylene glycol 8000, 0.15 µL of 100 mM dNTPs, 3 µL of 5X Maxima H RT Buffer, 0.2 µL of 200 U/µL Maxima H Reverse Transcriptase (ThermoFisher), 0.2 µL of 20 U/µL SUPERaseIN, and 0.15 µL of 100 µM Template Switching Oligo (Integrated DNA Technologies), and 1 µL of nuclease free water). Reverse transcription was performed at 42°C for 90 minutes, followed by 10 cycles of 50°C for 2 minutes, 42°C for 2 minutes, 75°C for 10 minutes, followed by a 4°C hold. Excess primers were removed by adding 2 µL of Exonuclease I mix (1.875U ExoI in water) to each well and incubating at 37°C for 30 minutes, 85°C for 15 minutes, 75°C for 30 seconds, 4°C hold. All wells were pooled into a single 15-ml falcon tubes and cDNA was purified and concentrated using Dynabeads™ MyOne™ Silane beads (ThermoFisher) according to the manufacturer's instructions. The cDNA was split into duplicate reactions containing 25 µl cDNA, 25 µl 2x HIFI HotStart Ready Mix (Kapa Biosystems), and 0.2 M SMART PCR Primer. PCR was run as follows: 37°C for 30 minutes, 85°C for 15 minutes, 75°C for 30 seconds, 4°C hold. Duplicate reactions were combined and purified using 0.7 volumes AMPure XP beads (Beckman Coulter). The amplified cDNA was visualized on an Agilent 2100 Bioanalyzer and quantified using a Qubit II fluorometer (ThermoFisher). Single-nucleus RNA-seq: NeuN nuclear sorting was conducted as described above. Approximately 70% NeuN+ and 30% NeuN- single nuclei were sorted per sample. Sorted nuclei were placed on ice and immediately submitted to the Columbia Single Cell Analysis Core (Columbia Genome Center) for 10X Genomics Chromium Single Cell 3' v3 library preparation according to the manufacturer's instructions. low-input bulk RNA-seq (PLATE-seq) and single-nucleus RNA-seq