single nuclei RNA-seq of Mus musculus: adult male mouse cortex
Cortical tissue was dissected from mice perfused with cold PBS buffer with a transcription inhibitor cocktail at a speed of 6 mL/min for 8 minutes and flash frozen in liquid nitrogen. The frozen tissue was subsequently placed into a tissue dounce for homogenization with homogenization buffer (0.25M sucrose, 25mM KCl, 5mM MgCl2, 20mM Tricine-KOH pH7.8 with freshly added 1mM DTT, 0.15mM spemine (Sigma), 0.5mM spermidine (Sigma), 0.04% BSA (Sigma) and RNase inhibitors (Promega)). 5% IGEPAL (Sigma) stock solution was added to the homogenization buffer to a final concentration of 0.32% for 20 strokes with tight pestle. The homogenate was visually inspected, passed through 40-m filter (BD Falcon) and layered onto an OptiPrep (Sigma) gradient, as previously described. The gradient was centrifuged at 10,000g for 18 minutes, and nuclei were collected between the 30% and 40% OptiPrep gradient layers. Single nucleic suspension was visually confirmed under microscope and diluted to 80,000 nuclei/mL for nuclei encapsulation and sequencing library preparation following an inDrop protocol as previously described130. Approximately about 1,500 nuclei per mouse cortex were encapsulated and passed the quality controls. The prepared libraries are sequenced on Illumina Nova-seq at Broad Institute Walk-up sequencing facility, at a coverage of about 7 billion reads for ~15,000 droplets (~450,000 reads per nuclei).