GSM7421529: fenster2020-TTCTAGCT; Macaca mulatta; RNA-Seq
After sacrifice, brains were rapidly dissected into slabs and frozen on metal plates in liquid nitrogen vapor. Time between animal sacrifice and tissue freezing was between 1-2 hours for all animals. Brains were kept stored at -80 C until punching, when they were punched on a microtome with guidance from a macaque anatomist (Dr. S. Haber). Punches were stored at -80 C until the day of nuclear isolation and encapsulation. Nuclei were isolated as in previous studies130, with minor modifications. Punches were placed into buffer HB (0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM Tricine-KOH pH 7.8, 1 mM DTT, 0.15 mM spermine, 0.5mM spermidine, protease inhibitors) and placed in a Dounce homogenizer for 10 strokes each with loose and tight pestles. A 5% IGEPAL solution was added to a final concentration of 0.3% followed by five additional dounce strokes, then the lysate was filtered through a 40-μm strainer. Nuclei were mixed with an equal volume of 50% iodixanol and then layered on top of an iodixanol gradient of 40% and 30% layers in a 2 mL dolphin microcentrifuge tube. Nuclei were spun by centrifuging at 10,000 x g for 4 min at 4°C and then collected by aspiration at the interface of the 30% and 40% iodixanol layers. Nuclear concentration and prep quality were ascertained by loading on a hemocytometer and were diluted to a concentration of 80-100K and 15% iodixanol with Buffer HB prior to loading on InDrops platform. Single-nuclei suspensions were encapsulated into droplets, lysed, and the RNA within each droplet was reverse-transcribed using a unique nucleotide barcode as described previously131. Approximately 6,000 nuclei, in two batches of 3,000 nuclei each were processed per library and sequenced on Illumina NovaSeq 6000 chips (at a density of approximately 20,000 reads/nucleus).