GSM9158850: GBS1, CD11b; Homo sapiens; RNA-Seq
Frozen cryovials containing 107 PBMCs/mL were thawed in a 37°C water bath for 2 minutes. Following thawing, 1 mL of pre-warmed RPMI 1640 medium (Gibco, 11875-093) supplemented with 10% HyClone™ Fetal Bovine Serum (Thermo Fisher Scientific, SH3008803IR) and 1% penicillin-streptomycin (Thermo Fisher Scientific, 15070063) was added to each vial. The cell suspension was transferred to a 15 mL conical tube, and the cryovials were rinsed with additional RPMI media to maximize PBMC recovery. To aid in cell dissociation, Accumax (Thermo Fisher Scientific, 00-4666-56) was added at a 1:1 ratio to the sample volume, and cells were incubated at room temperature for 5 minutes. Samples were centrifuged at 350g for 5 minutes at room temperature. The supernatant was aspirated, and the cell pellets were resuspended in 1 mL of RPMI media containing 100 µg/mL DNase I and incubated at 37°C for 15 minutes. Cells were washed with 10 mL of flow cytometry staining buffer (Invitrogen, 00-4222-26) and centrifuged at 350g for 5 minutes at room temperature. Cells were resuspended in 1X phosphate-buffered saline (PBS) and prepped for fluorescently activated cells sorting (FACS). First, cells were incubated with a Zombie UV viability stain for 10 minutes at room temperature. Then, 1 mL of flow cytometry staining buffer was added to each tube, cells were centrifuged, resuspended in flow cytometry staining buffer and treated with a human Fc receptor blocker for 10 minutes at room temperature protected from light. The cells were then treated with primary antibodies targeting CD45, CD3, CD4, CD8, and CD11b for 30 minutes, on ice, protected from light. Cells were then washed again with 1 mL of flow cytometry staining buffer, centrifuged, and resuspended in flow buffer and preserved on ice until sorting. FACS was performed on a BD FACSAria Fusion at the University of Texas at Dallas. Cells were gated to collect CD4+, CD8+, and CD11b+ cells in flow cytometry staining buffer. Tubes containing sorted cells were returned to the laboratory, centrifuged, resuspended in 1 mL of Qiazol (Qiagen, 1023537), transferred to a 1.5mL Eppendorf tube, and proceeded to RNA extraction. Next, 200 µL of chloroform (Fisher Scientific, C298-500) was added to each tube, manually shaken for 15 seconds, and allowed to incubate at room temperature for 2 minutes. Tubes were then centrifuged at 12,000g for 10 minutes at 4°C. The supernatant was collected, placed in a new 1.5 mL tube, and 500 µL of isopropanol (Fisher Scientific, A416-1) and 3 µg of GlycoBlue™ (Thermo Fisher Scientific, AM9515) were added. To maximize RNA precipitation, tubes were incubated overnight at -20°C. The next day, the cells were centrifuged at 12,000g for 30 minutes at 4°C. The supernatant was aspirated, and the resultant RNA pellet was washed with 1 mL of 75% ethanol (Fisher Scientific, BP2818-500). The tube was centrifuged at 7,500g for 5 minutes at 4°C and the supernatant was removed. The resultant RNA pellet was air dried for 10 minutes at room temperature and eluted in 20 µL of RNase-free water on a heat block at 55°C for 10 minutes. Sequencing was performed using a NextSeq2000 for single-end total RNA sequencing at the University of Texas at Dallas Genome Core Research Facility.