GSM9001054: Brain myeloid cells, Infected, GEX, rep1; Macaca mulatta; RNA-Seq
Animals were deeply anesthetized with ketamine plus xylazine, and blood was cleared from the brain and other organs by intracardial perfusion with sterile PBS containing 1 U/ml heparin. Brains were harvested, and approximately half of the brain was taken for microglia/macrophage isolation. Briefly, the brain was minced and homogenized in cold Hank's Balanced Salt Solution (HBSS, Invitrogen, Carlsbad, CA). After being centrifuged, the brain tissue was digested at 37° C in HBSS containing 28 U/ml DNase I and 8 U/ml papain for 30 minutes. After digestion, the enzymes were inactivated by adding 2.5% FBS, and the cells were centrifuged and resuspended in cold HBSS. The cell suspension was mixed with 90% Percoll (GE HealthCare, Pittsburg, PA) to a final concentration of 20% Percoll and centrifuged at 4° C for 15 minutes at 550 x g. The microglia/macrophage pallet at the bottom was resuspended in HBSS and passed through a 40 μm strainer to remove cell clumps and aggregates. Cells were again pelleted by centrifugation and resuspended in RBC lysis buffer for 3 minutes to eliminate contaminating red blood cells. A final wash was performed before the resulting cells were quantified on a hemocytometer and Coulter Counter Z1. The cells were resuspended in 10% DMSO and 90% fetal bovine serum and subjected to slow controlled freezing followed by storage in liquid N2. Nuclei were isolated from the cells and permeabilized as the 10XGenomics protocol. Cryopreserved cell isolates were rapidly thawed in a 37° C water bath. After the recovery, cells were washed and counted by Coulter Counter Z1. Once cell concentration was known, cells were transferred to ice-cold PBS and stained with CD11b (Biolegend 101257) and UV-blue live/dead (Invitrogen L23105). Cells were washed, resuspended in flow cytometry staining buffer (e-bioscience), and sorted on an Aria2 flow cytometer (BD Biosciences, San Jose, CA, USA). Nuclei were then quantified on a hemocytometer and concentrated to approximately 2200-2400 nuclei per µL. Based on 10× Genomics parameters targeting 8000 nuclei, the ideal volume of cells was loaded onto the 10× Genomics (Pleasanton, CA, USA) Chromium Next GEM Chip J and placed into the Chromium Controller for nuclei capturing and library preparation. The prepared libraries were sequenced using Illumina (San Diego, CA, USA) Novaseq6000 sequencers.
GEO
GSM9001054_r1