Chromium single cell 3 library and gel bead kit v3

Lungs were dissected from the embryo at the stages of interest and then dissociated with dispase (Corning) at room temperature for 10 min to generate a single-cell suspension. Cell suspensions were processed by the Princeton Genomics Core Facility. Cells were loaded and processed using the Chromium Single-Cell 3′ Library and Gel Bead Kit v3 (10x Genomics) on the Chromium Controller (10x Genomics) following the manufacturer’s protocols. Individual cells were encapsulated in droplets with individual gel beads carrying unique barcoded primers and lysed. Fragments of cDNA were synthesized, barcoded, and amplified by polymerase chain reaction (PCR). Illumina sequencing libraries were prepared from the amplified cDNA from each sample group using the Nextra DNA library prep kit (Illumina). Libraries were sequenced on NovaSeq 6000 S Prime flow cells (Illumina) as paired-end 28 + 94 nucleotide reads, following the manufacturer’s protocols. Base calling was performed, and raw sequencing reads were filtered using the Illumina sequencer control software to keep only pass-filtered reads for downstream analysis. The 10× Cell Ranger software version 3.0.2 was used to run the count pipeline with default settings on all FASTQ files from each sample to generate gene-barcode matrices using the Anolis carolinensis reference genome AnoCar2.0.