GSM8263313: PN, replicate 2, scRNA-seq; Mus musculus; RNA-Seq
Pontine nucleus tissue from two 8-week-old female C57BL/6J mice was microdissected, pooled, and flash frozen with dry ice. Tissues were Dounce homogenized with 500 microliters of chilled detergen lysis buffer (0.10% Triton-X, 0.32M sucrose, 10 mM HEPES [pH 8.0], 5mM CaCl2, 3mM MgAc, 0.1 mM EDTA, 1 mM dithiothreitol [DTT]) using 5 strokes with the "loose" pestle followed by 10 strokes of the "tight" pestle (Kimble). 1 mL of sucrose buffer (0.32 M sucrose, 10 mM HEPES [pH 8.0], 5 mM CaCl2, 3 mM MgAc, 0.1 mM EDTA, 1 mM DTT) was added to the Dounce homogenizer and the combined solution was passed through a 40-μm cell strainer into a fresh tube containing 1 mL of 0.32 M sucrose buffer. An additional 1 mL of 0.32 M sucrose buffer was passed through the filter and the resulting 3.5 mL solution was centrifuged at 3,200 xg for 10 min at 4°C. The pellet was resuspended with 3 mL of 0.32 M sucrose buffer and homogenized for 30 s (Ultra-Turrax disperser, setting 1). Next, 12.5 mL of 1 M sucrose buffer (1 M sucrose, 10 mM HEPES [pH 8.0], 3 mM MgAc, 1 mM DTT) was pipetted beneath the homogenate and the tube was centrifuged at 3,200 xg for 20 min at 4°C. After decanting the supernatant, the pellet was resuspended in 1 mL of resuspension solution (0.4 mg/mL BSA, 0.2 U/μL RNAse inhibitor [Lucigen] in 1X PBS), filtered through a 35-µm cell strainer, and diluted to a final concentration of 225 cells/µL. Single-nuclear suspensions were loaded onto two 10x Genomics chips (Chromium v3). snRNA-seq libraries were constructed according to the protocol provided by the manufacturer (Chromium single-cell 3' v3 protocol, 10x Genomics). Multiplexed snRNA-seq libraries were spiked with a PhiX control library (5%) and sequenced twice across two NextSeq 550 high-output flow-cell runs.