Lung tissue from wild type C57Bl/6J mice were isolated at sacrifice, quick frozen in liquid nitrogen, and stored at -80°C. Nuclei were isolated according to the instruction of nuclear separation kit (52009-10, SHBIO). Briefly, samples were cut to 7 mm pieces with 1 mL ice-cold nuclei lysis buffer. Tissue homogenizer grind tissues to liquid, then the suspension were passed through a 40 µm cell strainer to remove the impurity. Nuclei were centrifuged at 500 x g/5min, resuspended in lysis buffer, added reagents in turn to stratified. Sucked up the nuclear layer, and passed through a 40 µm cell strainer again. Resuspended nuclei and counted. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, nuclei were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the nuclei lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.