title: GSM8527120: SNr_DR_plate_5; Mus musculus; RNA-Seq
library_construction_protocol: Animals were euthanized in a CO2 chamber and decapitated, after which the brains were dissected out from the skull and submerged in ice-cold aCSF (92 mM NMDG, 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 5 Na-ascorbate, 3 mM Na-pyruvate, 0.5 mM CaCl2·2H2O, and 10 mM MgSO4·7H2O). Coronal brain slices (400 µm thick) were cut using a vibrating-blade microtome (V1200S, Leica, USA) at 4°C. The SNr subregion was microdissected out in aCSF under a fluorescent dissection microscope (Leica M165FC) and transferred to microcentrifuge tubes. Excess aCSF was aspirated before flash-freezing the tubes on dry ice. Single nuclei preparations were performed pooling tissue from 3-4 mice, following the Allen Institute's published V1 protocol. Briefly, each pooled sample was homogenized in a chilled 2 mL dounce (Sigma D8938) using 3.5 ml of freshly prepared homogenization buffer containing 3.375 ml of NIM (10 mM Tris-HCl, 250 mM sucrose, 25 mM KCl, 5mM MgCl2, 10mM Tris-HCl ), 3.5 µl of 100 mM DTT stock (Promega P1171), 70 µl of 50X Protease inhibitor cocktail (Promega G6521), 17.5 µl of 40 U/µl RNAsin Plus (Promega N2615), and 35 µl of 10% Triton X-100 (Sigma T8787). The homogenate was strained through a 30 µm cell strainer (Miltenyi Biotec 130-098-458) and centrifuged at 900 x g for 10 minutes at 4°C. After centrifugation, the pellet was resuspended in 500 µl of freshly prepared blocking buffer containing 410 µl nuclease-free water, 50 µl 10X PBS, 40 µl of 10% OmniPur BSA (Sigma 2905), and 2.5 µl of 40 U/µl RNAsin Plus. The nuclear resuspension as subsequently incubated on ice for 15 minutes, , then labeled with 0.5 µl of DAPI. Prior to DAPI labeling, 5 µl of nuclear suspension was removed and labeled with 5 µl of Trypan Blue and loaded into a disposable hemocytometer slide (Invitrogen C10228) for quantification of yield and visual inspection of nuclear quality. Fluorescence-activated nuclei sorting (FANS) of single nuclei was performed using the Beckman Coulter MoFlo Astrios EQ. Cell analysis and sorting were performed on a Beckman Coulter MoFlo Astrios EQ. A 405 nm (80 mW) laser was used for excitation of DAPI and a 561 nm (200 mW) laser was used for excitation of mCherry. DAPI was detected with a 448 /59 band-pass filter. mCherry was detected with a 614 /20 band-pass filter. DAPI+/mCherry+ nuclei were collected and sorted into 96-well plates preloaded with a lysis solution (6 µl per well) including 0.2% Triton X-100, 1 U/ul SuperaseIN (Invitrogen), 1 mM dNTPs and RT primer. Individual nuclei were sorted into the 96-well plates on “Single Cell” mode with a drop envelope of “1”. Sheath pressure was set at 28 PSI.  Sample pressure was kept below 28.5 PSI.  A 100 µm nozzle was used.  Cells were run at an event rate between 2000 – 5000 cells / second. For cell sorting experiments a droplet drive frequency of 49.6 KHz was used. After sorting, 96-well plates were briefly centrifuged, flash frozen on dry ice and stored at -80°C prior to library preparation. Primer annealing was performed by incubating plates on a thermocycler at 72°C for 3 minutes, followed by hold at 4°C. A reverse transcription reaction mix (7.5 µl) was added to each well. Each 7.5 µl of reaction mix contained 40U Maxima H Minus Reverse Transcriptase (ThermoFisher), 4U SupernaseIN, 7.5% PEG and 1uM TSO and 1X Maxima Buffer. Reverse transcription was performed by incubating plates on a thermocycler at 42°C for 90 minutes, followed by 10 cycles of 50°C for 2 minutes and 42°C for 2 minutes. Reverse transcription was stopped by heating at 75°C for 10 minutes, followed by hold at 4°C. An ExoI digestion mix (0.22 U/ µl) was subsequently added to each well to remove excess primer or singled stranded products. ExoI digestion was performed by incubating plates on a thermocycler at 37°C for 30 minutes, 85°C for 15 minutes and 75°C for 30 seconds, followed by hold at 4°C. Pooling was performed on the Biomek 4000 (Beckman Coulter, Brea California). cDNA cleanup was performed using Silane beads (ThermoFisher) suspended in RLT Plus buffer (Qiagen) and ethanol. Bead incubation was performed at room temperature for 20 minutes followed by placement on a magnetic stand. The supernatant was removed and the beads were washed twice with 80% ethanol before being returned to the magnet and allowed to air dry for 10 minutes. The beads were resuspended in 50 µl of nuclease-free water, incubated for 2 minutes and returned to the magnet. The supernatant was then removed, transferred to a clean Eppendorf tube and then split into two tubes for cDNA amplification. cDNA amplification was performed using a master mix containing 25 µl of 2X Kapa HotStart Mix (Roche) per reaction and 2 µl of 5 µM SMART PCR primer (Takara Bio). Amplification was performed by incubating tubes on a thermocycler at 98°C for 3 minutes followed by 18 cycles of 98°C for 20 seconds, 67°C for 15 seconds and 72°C for 5 minutes. The reaction was then maintained at 72°C for 5 minutes, followed by hold at 4°C. The two reactions were pooled in one Eppendorf tube and 70 µl of AMPureXP beads (Beckman Coulter) were added to the sample and incubated for 5 minutes at room temperature. The sample was placed on a magnet and the supernatant was removed. The beads were then washed twice with 80% ethanol, returned to the magnet and dried for 5 minutes. The beads were then resuspended in 25 µl of nuclease-free water, incubated for 2 minutes and placed on the magnet for removal of the supernatant to a new tube. The quality and quantity of cDNA was examined using an Agilent Bioanalyzer and Qubit Fluorometer (ThermoFisher). Sequencing libraries were prepared using Nextera XT Library Prep Kit (Illumina). 0.6 ng of cDNA was used as input and tagmentation and neutralization was performed as directed. Indexing PCR was performed as directed using 10 cycles by adding 5 µl of 2 µM custom i5 primer, 5 µl N7 Nextera primer and 15 µl of NPM. PCR was followed by 2 AMPureXP cleanups as above, first with 0.6 beads:sample ratio and then with 1:1 beads:sample. The final volume was eluted in 20 µl of nuclease-free water. QC was repeated as before. Libraries were sequenced using an Illumina NextSeq 500/550.