GSM9634794: non-diseased control_2_ADT; Homo sapiens; OTHER
Tissue samples were obtained from fibrotic lungs [Idiopathic Pulmonary Fibrosis (IPF), Interstitial Pneumonia with Autoimmune Features (IPAF), and Connective Tissue Disease-associated Interstitial Lung Disease (CTD-ILD), Nonspecific Interstitial pneumonia (NSID)] removed at the time of lung transplant surgery or non-fibrotic control lungs (declined donors) obtained at the time of organ procurement at Vanderbilt University Medical Center. Lung tissue biopsies were washed in PBS, minced, and frozen in RPMI media containing 10% DMSO until analysis. Minced lungs were thawed at 37oC for 3 minutes and digested in an enzymatic cocktail [Collagenase XI and DNAse I, type IV (Sigma)] and using a gentleMACS Octo Dissociator (Miltenyi Inc.) at 37°C and filtered using 100-μm sterile filters (Fisher). Whole lung cell suspensions (up to 1 million cells) were incubated with an Fc receptor blocking reagent (TrueStain FcX, BioLegend, USA) for 15 minutes, followed by staining with a panel of flow cytometry antibodies [anti-CD45 (APC-Cy7), anti-CD3 (PE-Cy5), and fourteen oligo-conjugated TotalSeq-C human antibodies (BioLegend)] for 30 minutes. Cells were washed three times with PBS containing 1% BSA and stained with a viability dye (Zombie Aqua kit, BioLegend). Up to 25,000 viable CD45⁺CD3⁺ cells were sorted per sample using a BD FACS ARIA and submitted for sequencing on the 10x Genomics Chromium platform. Briefly, 10,000 cells were loaded onto a 10x Chromium single-cell encapsulation chip, and libraries were prepared according to the manufacturer's instructions. Sequencing was performed on Illumina platforms at a depth of at least 80 million reads per library. 10x_Single Cell 5' PE