Single cell RNA-seq atlas of human NSCLC lesions and non-involved tissue
Single cell RNA-seq atlas of human NSCLC lesions and non-involved tissue
Tissues were kept in cold complete RPMI medium (RPMI [Invitrogen] supplemented with  10% FBS [Sigma Millipore], 2mM L-Glutamine [Life Technologies] and 100U/ml Penicillin-Streptomycin [Thermofisher]) until dissection, which was performed on the same day of collection. Single-cell suspensions were generated as previously described25. Briefly, tissues were placed into a petri dish and cutted into small pieces of 2-4 mm and transferred into a 1.5ml tube containing the digestion mix (complete RPMI media supplemented with  1 mg ml−1 collagenase IV and 0.1 mg ml−1 DNase I) and minced using surgical scissors. Minced tissues were incubated for 45 minutes at 37℃ and vortexed every 15 minutes. Digested tissues were passed through a 100 μm strain into a falcon tube prefilled with cold PBS.  Cells were then centrifuged for 5 minutes at 300 g, 4°C and the pellet was resuspended into 1x RBC lysis buffer (eBioscience) for 2 minutes at room temperature, after which 20 ml of cold PBS were added to stop the lysis reaction. Cells were cryopreserved in 5% DMSO in KnockOut Serum Replacement (KOSR; GibcoTM) until further use.  On the day of the experiment, cells were rapidly thawed at 37℃ and transferred to complete RPMI media. Live cell enrichment was performed using MACS Dead Cell Removal Kit (Miltenyi Biotec) following the manufacturer's instructions. Red blood cells were further depleted by negative selection using CD235a Microbeads (Miltenyi Biotec) and MACS LS columns (Miltenyi Biotec), following the manufacturer's instructions.  For FACS sorting, ce​lls were stained with Zombie Aqua to exclude dead cells and the cocktail of antibodies  for 30 minutes at ​4°C. Cells were centrifuged for 5 minutes at 300 g, 4°C, resuspended in 500 μl of 5% FBS in PBS and subsequently filtered into polypropylene FACS tubes.   Immune  cells were sorted as live, CD45+; MDSC were sorted as live, CD45+, Lineage- (Lin: CD3, CD56, CD19), CD11b+, CD33+;. e-MDSC were sorted as live, CD45+, Lin-, CD33+, HLA-DR-/low. Cells were sorted into a 1.5 ml tube, counted and submitted for 10x scRNA seq library preparation. Each cell suspension was submitted for 3' single cell RNA sequencing using Single Cell G Chip Kit, chemistry v3.1 (10x Genomics Pleasanton, CA, USA), following the manufacturer's instructions.  Each cell suspension was submitted for 3' single cell RNA sequencing using Single Cell G Chip Kit, chemistry v3.1 (10x Genomics Pleasanton, CA, USA), following the manufacturer's instructions.