GSM7295760: KT, replicate 2, ATAC-seq 2; Mus musculus; ATAC-seq
KT, KFT, and KPT mice were transduced with Ad5-SPC-Cre and harvested for cell sorting at 10 weeks post-transduction. Non-transduced lungs were used as a control. Briefly, lungs were perfused with 10 mL of ice-cold PBS and placed in a sterile dish on ice. Lungs were finely minced with sterile scissors and razor blades and resuspended in 6 mL digestion media (RPMI with 2.5 mg of Liberase TL (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 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). ATAC-sequencing was performed as previously described. Two independent ATAC-sequencing experiments were performed, one with KT and KPT mice and the second with KT and KFT cells. Briefly, 5x105 cells were sorted and washed 2x in FACS buffer without DNase added. Nuclei were then isolated, lysed, and incubated with Tn5 transposase precisely as described. Transposed DNA was then isolated using the MinElute Reaction Cleanup Kit (Qiagen). Libraries were prepared by amplifying DNA for 5 cycles with NEBNext 2x MasterMix (NEB), after which qPCR was run on samples to determine the number of additional cycles needed. After any additional amplifications were performed, libraries were purified using the MinElute Reaction Cleanup Kit (Qiagen). Samples were sequenced on a HiSeq4000 (Illumina) (KT vs. KPT experiment).