GSM9096776: BALF immune cells, 3 hours 1 ppm Ozone-exposed; Mus musculus; RNA-Seq
mice were anesthetized via intraperitoneal injection of 2,2,2-tribromoethanol (Millipore Sigma, Burlington, MA). Midline laparotomy was performed to expose and severe inferior vena cava for exsanguination. Thereafter, thoracotomy was performed to expose the lung and trachea. The left main stem bronchus was ligated, and the right lung lobes were lavaged with a calculated volume of ice-cold Dulbecco's Phosphate Buffered Saline (DPBS) (Corning, Manassas, VA). The retrieved bronchoalveolar lavage fluid (BALF) was centrifuged at 500 x g for 5 min at 4°C. To maximize the recovery of airspace immune cells, further lavages were performed to collect an additional 5 ml of BALF. The remaining portion of the immune cells from the first lavage and the additional 5 ml lavage of 3-4 mice per group were pooled and centrifuged at 500 x g for 5 min at 4°C. The supernatant was removed, and the cell pellet was resuspended in 500 ml of fresh DPBS. The washing process was repeated one more time. After the last wash, the BALF was centrifuged at 500 x g for 5 min at 4°C, the supernatant was removed, and the cell pellet was resuspended in DPBS with 1% Fetal Bovine Serum (FBS). The concentration of BALF immune cells was adjusted to 2000 cells/ml. Samples were submitted to the Advance Analytics Core at the University of North Carolina at Chapel Hill (UNC) School of Medicine for library construction and sequencing. BALF immune cells collected from C57BL/6J mice were stained with acridine orange and propidium iodide and assessed for viability and concentration using the LUNA-FX7 dual fluorescence cell counter (Logos Biosystems, Annandale, VA), and loaded into a Chromium Next GEM Chip G (10X genomics, Pleasanton, CA) to generate gel-beads in emulsions. Barcoded sequencing libraries were conducted following the instruction manual of the Chromium Single Cell 3' Reagent Kit v3.1 (10X genomics, Pleasanton, CA). Single-cell libraries were sequenced using NovaSeq X Plus (Admera Health, South Plainfield, NJ).