title: GSM7965613: Clec5a_KO_CLP_2; Mus musculus; RNA-Seq
library_construction_protocol: The lung tissue was placed in a sterile RNase-free culture dish containing an appropriate amount of calcium-free and magnesium-free 1 × PBS on ice and cut it into pieces, and then they were washed with 1 × PBS and remove non-purpose tissues. Tissues were dissociated into single cells in dissociation solution (0.35% collagenase IV5,2 mg/ml papain, 120 Units/ml DNase I) in 37 ℃ water bath with shaking for 20 min at 100 rpm. Digestion was terminated with 1× PBS containing 10% fetal bovine serum（FBS，V/V）, and the resulting cell suspension was filtered and centrifuged at 300g for 5 min at 4 °C. The cell pellet was resuspended in 100ul 1× PBS (0.04% BSA) and added with 1 ml 1× red blood cell lysis buffer to lyse remaining red blood cells. After incubation, the suspension was centrifuged at 300g for 5 min at room temperature. The suspension was resuspended in 100 μl Dead Cell Removal MicroBeads and remove dead cells. Then the suspension was resuspended in 1× PBS (0.04% BSA) and centrifuged at 300 g for 3 min at 4 °C（repeat twice）. The cell pellet was resuspended in 50 μl of 1× PBS (0.04% BSA). The overall cell viability (above 85%) was confirmed by trypan blue exclusion, and single cell suspensions were counted and concentration adjusted to 700-1200 cells/μl. Single-cell suspensions were loaded to 10x Chromium to capture 5000 single cell according to the manufacturer's instructions of 10X Genomics Chromium Single-Cell 3'kit (V3).The following cDNA amplification and library construction steps were performed according to the standard protocol. Libraries were sequenced on an Illumina NovaSeq 6000 sequencing system (paired-end multiplexing run, 150bp) by LC-Bio Technology co.ltd. at a minimum depth of 20,000 reads per cell.