GSM8884912: 92T_brain_CD45,non-infection,rep2; Macaca mulatta; RNA-Seq
For isolating brain lymphocytes, a necropsy was performed on deeply anesthetized (ketamine plus xylazine) animals, following intracardial perfusion with sterile PBS containing 1 U/ml heparin. Brains were harvested, and approximately half of the brain was taken for cell isolation.The brain was minced and homogenized in cold Hank's Balanced Salt Solution (HBSS, Invitrogen, Carlsbad, CA, USA). 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 brought to a final concentration of 20% Percoll by the addition of 90% Percoll (GE HealthCare, Pittsburg, PA, USA) and centrifuged at 4° C for 15 minutes at 550 x g. The cell pallet at the bottom was resuspended in HBSS and passed through a 40 μm strainer to remove cell clumps and/or aggregates. Cells were again pelleted by centrifugation. A final wash was performed before the resulting cells were quantified on a hemocytometer and Coulter Counter Z1.The brain cells were cryopreserved before the cell sorting and scRNA-seq. To isolate the brain lymphocytes, samples of cryopreserved brain cell isolates, stored in liquid nitrogen , 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, staining with Live/Dead and PE-labeled anti-CD45 antibody (Biolegend, San Diego, CA). The live cells that were CD45+ were collected for scRNA-seq capture and library preparation. For isolating blood CD4+ lymphocytes, freshly isolated EDTA-anticoagulated blood was centrifuged, and the cells washed with PBS and then stained with UV-blue live/dead for 30 minutes at 4° C. After being blocked with FACS buffer for 3 minutes, the cells were centrifuged and resuspended in the antibody cocktail mixed in Brilliant Stain buffer (Catalog# 563794, BD Biosciences, San Jose, CA, USA) for 45 minutes. After incubation, red blood cell (RBC) lysing buffer was added to the sample and the cells were pelleted at 300 x g for 8 minutes. After centrifugation, the cells were resuspended in RBC lysing buffer, which was repeated to completely remove RBCs. The cells were then pelleted and resuspended in MACS buffer for FACS sorting. The antibody cocktail included V500-labeled anti-CD3 antibody (Catalog# 561417, BD Biosciences, San Jose, CA, USA), BV786-labeled anti-CD4 antibody (Catalog# 563881, BD Biosciences, San Jose, CA, USA), PE-CF594-labeled anti-CD20 antibody (Catalog# 562295, BD Biosciences, San Jose, CA, USA) The selection of cells was based on the size, singlet, live, and expression of CD20, CD3, and CD4. The CD20-positive cells were excluded, and the CD20-negative cells that were positive for CD3 and CD4 were collected for scRNA-seq library preparations.  For cell cocultivation experiment, MDM or MDM-jurkats cocultivation system was harvested by incubating with trypLE (Catalog# 12605-010, Gibco, Grand Island, NY, USA)  for 10 minutes at 37 ℃, then after one step of washing with 1mL PBS, another 1 mL PBS (Catalog# 14190-136, Gibco, Grand Island, NY, USA) with 0.04% BSA (Catalog# 130-091-376, Miltenyi Biotec, Bergisch Gladbach, Germany) was added for scraping the cells from the plate. For the cocultivation groups,  the harvested cells were directly used for library preparations. For the non-cocultivation group, the MDM was mixed with jurkat cells for library preparations. Post-sorting, isolates were concentrated to approximately 1000 cells per µL and assessed by trypan blue for viability and concentration. Brain immune cells and blood CD4+ T cells are isolated from the acutely infected macaques that were processed using the 10× Genomics (Pleasanton, CA, USA) Chromium 3' v3 GEM kit targeting 8000 cells. And cocultivation study used 10× Genomics GEM-X Universal 3' Gene Expression v4 4-plex kit targeting 5000 cells. Based on the targeting cell number, the ideal volume of cells was loaded onto the 10× Genomics Chromium GEM Chip and placed into the Chromium Controller for cell capturing and library preparation. In short, this occurs through microfluidics and combining with Single Cell 3' Gel Beads containing unique barcoded primers with a unique molecular identifier (UMI), followed by lysis of cells and barcoded reverse transcription of RNA, amplification of barcoded cDNA, fragmentation of cDNA to 200 bp, 5' adapter attachment, and sample indexing as the manufacturer instructed with version 3 reagent kits. The prepared libraries were sequenced using Illumina (San Diego, CA, USA) Nextseq550 and Novaseq6000 sequencers.