title: GSM5179461: LHA sample pool 2; Mus musculus; RNA-Seq
library_construction_protocol: C57BL/6J mice (n = 6, 3 male/3 female, 6-8 weeks old at time of surgery) were injected bilaterally with retroAAV2-hSyn-eYFP in LHb and retroAAV2-CAG-tdTomato in VTA (300-400 µL).  Approximately six weeks later, the mice were deeply anesthetized with sodium pentobarbital and phenytoin sodium and transcardially perfused with 10 mL ice-cold NMDG-aCSF containing 96 mM NMDG, 2.5 mM KCl, 1.35 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 2 mM thiourea, 5 mM Na+ascorbate, 3 mM Na+pyruvate, 0.6 mM glutathione-ethyl-ester, 2 mM N-acetyl-cysteine, 0.5 mM CaCl2, 10 mM MgSO4; pH 7.35–7.40, 300-305 mOsm. Six 280 µm sections through the LHA were collected in NMDG-aCSF on a vibratome. Sections recovered in NMDG-aCSF containing 500 nM TTX, 10 μM APV, 10 μM DNQX (NMDG-aCSF-R) at room temperature for 40 minutes. After recovery, LHA tissue was isolated with tissue punches and incubated in NMDG-aCSF-R containing 1.0 mg/mL pronase for 50 minutes at room temperature. Following digestion, tissue from all animals was transferred to 1.0 mL NMDG-aCSF-R supplemented with 0.05% BSA (NMDG-aCSF-BSA), pooled, and mechanically dissociated with a patch pipet fire-polished to an internal diameter of 200-300 μm. The suspension was washed in 12 mL NMDG-aCSF-BSA, sedimented at 220 xg for 6 minutes at 18ºC. Dead cells and cellular fragments were removed using a MACS® Dead Cell Removal Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) using 100 μL of dead cell removal beads and one MACs LS column following the standard manufacturer protocol. Following the final wash, cells were sedimented at 220 xg for 6 minutes at 4ºC and resuspended in PBS containing 0.05% nuclease-free BSA for a final concentration of 700-800 cells/μL. Single-cell RNA-sequencing was performed using Chromium Single Cell 3' v3 chemistry (10X Genomics, Pleasanton, CA) following manufacturer recommendations unless otherwise noted. Approximately 16,000-18,000 pooled cells were loaded into an individual well of a Single Cell B Chip, with three wells used in total for three reactions. cDNA amplification and sample indexing were each performed using 11 cycles. Library size distribution and concentrations were determined with an Agilent 4200 Tapestation High Sensitivity D5000 ScreenTape and a Thermo Scientific Qubit dsDNA HS Assay, respectively. Each reaction library was sequenced on an individual lane of an Illumina HiSeq 4000 using 2x150 chemistry following the standard 10X Genomics v3 paired-end configuration by Genewiz, Inc. (South Plainfield, NJ). Between approximately 370-430 million reads were generated per lane with a mean quality score >32 across all lanes.
attributes.tag: GEO Accession
attributes.value: GSM5179461