single-cell RNA-seq of mus musculus: adult female lung
The female BALB/c mice (6-8-weeks old) were immunized twice with CNP (n=4 per group) and mice were sacrificed at 21 days post prime vaccination. The lungs were harvested and minced with scissors, following treated with collagenase I (1 mg/mL) and DNase I (0.05 mg/mL) and dissociated to a single-cell suspension. After treated with red blood cell lysing reagent, the single-cell suspension was passed through a 40 m strainers and resuspended in PBS. The single-cell suspension of four mice in each group were pooled. The CD45+ cells were isolated from single-cell suspension in each group by CD45 MicroBeads (130-052-301, Miltenyi Biotec), and then these CD45+ single-cell suspensions that containing about 10000 cells in each group were loaded on a 10X Genomics GemCode Single-cell instrument to generates single-cell Gel Bead-In-EMlusion (GEMs). Libraries were generated and sequenced from the cDNAs with Chromium Next GEM Single Cell 3 Reagent Kits v3.1. The Gene Denovo Biotechnology Co. (Guangzhou, China) were entrusted to perform GEMs generation, cDNA amplification, library preparation and sequencing in this study.