design: Transcriptomic profiling of immune and stromal cells from the non small cell lung cancer (NSCLC) environment
library_construction_protocol: Samples were collected after surgical resection of lung lobe or wedges from patients. Fresh surgical specimens of NSCLC and unaffected autologous lung tissue were mechanically disaggregated and tissue was digested using a MACS human tumor dissociation kit (Miltenyi Biotec) according to the manufacturer's protocol. Hematopoietic, erythroid and tumor cells were depleted by incubating the cell suspension with MACS anti-CD45, anti-CD235a (Glycophorin A) and anti-CD326 (EpCAM) MicroBeads (Miltenyi Biotec) and passing through a MACS LS column (Miltenyi Biotec) to enrich for stromal cells. The flow-through was centrifuged and single cell suspension was stained for further flow cytometric analysis or cell sorting. The unbound single cell suspension was stained for cell sorting with a BD FACS Melody 2-way or 4-way cell sorter (BD Biosciences) and run on the 10x Chromium analyzer (10X Genomics). For the isolation of immune cells, tissues were gently smashed through a mesh using a syringe plunger and washed with RMPI1640 containing 2% FCS and 20 mM HEPES (Sigma-Aldrich) before staining for flow cytometric analysis or cell sorting. cDNA library generation was performed following the established commercial protocol for Chromium Next GEM Single Cell 3ʹ GEM, Library & Gel Bead Kit v3/3.1 Chemistry.