GSM8062770: Occipital Cortex, NHP2; Macaca mulatta; RNA-Seq
For nucleus isolation, buffers were typically prepared as described below, cooled in advance, and then supplemented immediately prior to use to make “complete” buffers at a final concentration of 1 mM dithiothreitol, 0.8 U/µl RNase inhibitor (Protector RNase Inhibitor; Roche, Indianapolis, IN, USA), and 1X protease inhibitor (complete Mini EDTA-free, Roche).  For these assays ~25 mg of frozen tissue was minced with a scalpel and transferred to a pre-chilled 2-mL Dounce homogenizer with 1 mL of cold complete lysis buffer (0.32 M sucrose, 5 mM CaCl2, 3 mM magnesium acetate, 0.1 mM EDTA, 10 mM Tris-HCl pH 8.0, and 0.1% Triton X-100). The tissue was homogenized with 10 strokes each of pestle A then B and sequentially passed through pre-wetted 100-µM and 30-µM filters, with the filtered sample collected in a sterile tube. We washed the homogenizer and filters with an additional three volumes of complete lysis buffer, collected with the filtered sample. Two volumes of sample were then layered on top of one volume of cold complete isolation buffer (1.8 M sucrose, 3 mM magnesium acetate, and 10 mM Tris-HCl pH 8.0) and spun at 21 000g for 45 min at 4°C.  We then carefully removed the supernatant and added 100 µL complete resuspension buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, and 20 mM Tris-HCl pH 7.2) to the tube without mixing. We incubated the samples on ice for 10–15 min, fully resuspended the nuclei by gently pipetting up and down 20–30 times, and then counted the nuclei with an automated cell counter (Countess 3, Thermo Fisher). For submission of single-nucleus transcriptomic samples, we adjusted the concentration to ~1×106 nuclei/mL (range of 0.7–1.2×106 nuclei/mL) as per the manufacturer's sample-loading guidelines. Library preparation was performed according to the manufacturer's instructions (Single Cell 3' v3.1 protocol, 10x Genomics). Briefly, a single nuclei suspension was loaded alongside a master mix and gel beads. The gel beads and individual nuclei were partitioned into droplets to generate an emulsion. A unique molecular identifier and cell barcode were linked to poly-A RNA and reverse-transcribed into cDNA. The cDNA was recovered from the emulsion using silane Dynabeads, amplified to generate sufficient material for library preparation, and then size selected and purified post-amplification with SPRI-Select beads. A portion of the purified cDNA was then enzymatically fragmented, and partial Illumina adapters were ligated. A final PCR amplification further enriched the libraries and completed the Illumina adapters for sequencing, incorporating a unique sample index for demultiplexing