GSM6758159: Homeostasis CD45+ replicate 1; Mus musculus; RNA-Seq
Mice were euthanized and anti-CD45 antibody was instilled into the trachea. After 4 minutes, anti-CD45 solution was aspirated and bronchoalveolar lavage (BAL) was performed. Lungs were perfused by injecting 10 mL of PBS through the right ventricle during which lungs visibly blanched. Lung tissue was finely chopped with a razor blade and then incubated at 37C in 1 mL of 0.4 mg/mL Liberase TM for 25 min. Following incubation, tissue was pipetted rapidly up and down to further disaggregate, then filtered through 100 μm filters. Lung cells were pelleted and resuspended in then filter through 70 μm filters then resuspended HBSS containing 0.3% BSA and 0.3 mM EDTA. Cells were sorted using FACS into 3 bins: 1) CD45+ cells (live, CD45+), 2) resident IMs (live, CD45+, CD64+, CD88+, IT CD45-, tdT+), or 3) recruited IM (live, CD45+, CD64+, CD88+, IT CD45-, tdT-). Cells were manually counted using a hemocytometer and resuspended at 1x 10^6 cells/ml in 0.06% BSA in PBS solution. Library was contructed according to manufacturer's instructions. Single cells were captured using a Chromium Box (10x Genomics, Pleasanton, CA, USA). Libraries were sequenced using the Illumina NovaSEQ 6000 sequencer (Illumina, Inc., San Diego, CA).  Sequencing was targeted to 5,000 cells in the CD45+ library, 3,000 cells in each of the IM sorts with sequencing depth of 100,000 reads per cell.