GSM8030991: Replicate 1, macaque, snRNA-seq; Macaca mulatta; RNA-Seq
Three male macaques (3-years old) were used for the single nuclear RNA-sequencing. Snap-frozen lumbar spinal cord segments were removed from -80ºC storage and placed into separate Petri dishes containing a cold slurry of dissection buffer consisting of 1x Phosphate Buffered Saline (PBS, ThermoFisher Scientific; AM9625), 10% Dithiothreitol (DTT, Sigma; 43816-50ML). From each animal, transverse sections (~50-75 mg) were made using a sterile razor blade. Three sections were selected for further dissection and the rest were discarded. The sections were cut in the coronal plane at the middle of the spinal cord to obtain dorsal and ventral halves. Any residual meningeal membranes were removed. The three dorsal halves were retained and the ventral halves discarded. Nuclei were isolated according to Martelotto with modifications73. The dorsal halves for each animal were transferred into separate Dounce homogenizers (Sigma) containing 1 mL of ice-cold homogenization buffer consisting of EZ Nuclei Lysis Buffer (Sigma; NUC101-1KT) with 0.5% RNasin Plus (Promega; N2615), 0.5% SUPERase-In (ThermoFisher; AM2696) and 1mM DTT. The samples were homogenized on ice using 20 strokes of Pestle A followed by 20 strokes of Pestle B. Any residual meningeal membrane was removed before switching pestles. The homogenate was filtered through a 50 µm filter (Sysmex; 04-004-2327) into a 2 mL microcentrifuge tube (Eppendorf; 022431048). An additional 0.5 mL of homogenization buffer was used to wash the Dounce homogenizer and filter. The sample was then placed on ice while the remaining samples were processed. The sample was centrifuged at 500g at 4ºC for 5 min to obtain a crude pellet containing spinal nuclei. The supernatant was removed and discarded, being careful to not disturb the pellet. The pellet was resuspended in 1.5 mL of Homogenization Buffer and allowed to sit on ice for 5 mins. The samples were again centrifuged at 500x g, 4ºC for 5 min. The supernatant was removed and the pellet was resuspended in 1 mL of Nuclei Resuspension Buffer (NRB) consisting of 1x PBS, 1% Bovine Serum Albumin (BSA, Sigma; 2905-5GM) and 1% SUPERas-In followed by centrifugation at 500g, 4ºC for 5 mins. This wash step was repeated twice more for a total of 3 washes. The final pellet was resuspended in 0.5 mL of NRB containing 6 µM 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI, ThermoFisher; D1306). The suspension was filtered through a 20 µm filter (Sysmex; 04-004-2325) into a polypropylene tube and kept on ice. Fluorescence Activated Nuclear Sorting (FANS) was performed to purify nuclei from debris on a FACSAria II (BD). Gates were set to isolate DAPI+ singlet nuclei based on forward scatter and side scatter as well as fluorescence intensity. The instrument was set to 40 pounds per square inch (psi) of pressure and a 70 μm nozzle was used, with sterile PBS sheath fluid. Nuclei were sorted into a 1.5 ml microcentrifuge tube containing 15 μl of NRB at 4ºC. For each sample, 18,000 events were sorted into the collection tube. The sorted nuclei and NRB total volume were approximately 45 µl, allowing for loading the entire suspension into the Chromium Single Cell 3' v3 solution (10x Genomics) without further manipulation. The 10x libraries were processed according to the manufacturer's instructions. Completed libraries were run on the Novaseq 6000 (Illumina). Library construction and sequencing were done at the 10X Genomics Core at the University of Pittsburgh according to standard 10x workflow: https://www.10xgenomics.com/support/single-cell-atac/documentation/steps/library-prep