GSM7295725: KFT, replicate 2, 10x multiome ATAC-Seq; Mus musculus; ATAC-seq
KFT, KT, and KPT mice were treated with Ad5-SPC-Cre as described above. After 10 weeks, lungs were harvested and were perfused with 10 mL of ice-cold PBS and placed in a sterile dish on ice. Lungs were minced with sterile scissors and resuspended in 6 mL digestion media (RPMI with 0.083 U/mL of collagenase type IV (Sigma-Aldrich), 0.6 U/mL dispase ii (Sigma-Aldrich), and 25 µg/mL DNase (Sigma Aldrich)).  Lungs were rotated for 45 minutes at 37ºC. Tubes were briefly cooled on ice, and then samples were passed through a 70 µM filter followed by a 40 µM filter. 5 mL of FACS buffer (10% FBS, 2 mM EDTA, 25 µg/mL DNase in DPBS) was added and tubes were spun for 5 minutes at 300g. Cells were resuspended in ACK lysing buffer (Gibco), incubated for 1 minute on ice, and quenched with 8 mL of FACS buffer. Cells were washed 2x with FACS buffer, resuspended with biotinylated primary antibodies (CD45, BioLegend 103104 30-F11; CD31, BioLegend 102404 390; F4/80, BioLegend 123106 BM8; Ter119, BioLegend 116204 TER-119, 1:800 in FACS buffer) and incubated for 20 minutes on ice. After washing 2x, cells were resuspended in streptavidin-APC secondary antibody (BioLegend 405207, 1:800) for 20 minutes on ice. Cells were washed 2x and resuspended in FACS buffer with 1 µg/mL DAPI, filtered through a 40 µM filter, and sorted using a Sony SH800S cell sorter. Data were analyzed using the default Sony SH800S software and FCS Express (Version 7, De Novo Software). Single cell suspensions were prepared from mice with lung tumors as described above. Nuclei were isolated and libraries were prepared using the 10x Genomics Chromium Single Cell Multiome ATAC + Gene Expression kit according to the manufacturer's protocols. An input of at least 2,000 nuclei were added to each 10x channel. Libraries were sequenced on an Illumina NovaSeq6000. 10x multiome ATAC