GSM7295190: Lung immune cell, scRNAseq; Homo sapiens; RNA-Seq
Freshly collected lung specimens of the paraquat poisoning patient were obtained from the lung removed at the time of lung transplantation. For primary cell isolation, the lung specimens without mesothelial layer were minced mechanically into small pieces (<1 mm3) and digested in modified DMEM medium with an enzyme cocktail containing 10 U/mL neutral protease, 400 U/mL collagenase type I, 8 U/mL elastase, and 0.66 U/mL DNase I for 50 mins at 37°C. To stop the enzymatic reaction, the digested tissues were added 10% FBS and incubated for 10 mins at RT. Digested tissue was filtered using 100 m- and 40 m-cell strainer. The filtrated suspension was centrifuged at 400 g at 4°C for 8 mins to collect the dissociated cells. The cell pellet was resuspended in RBC lysis buffer and incubated for 2 mins at RT to lyse the red blood cells. After centrifugation, the cell pellet was resuspended in freezing DMEM+5% FBS to stain with PE anti-human EPCAM, FITC anti-human CD45, APC anti-human CD31 at 4 °C for 30 mins. The cell pellet was resuspended with DMEM+2% FBS and filtered through a 40 m cell strainer again before the sorting process. DAPI was used to label dead cells. Sorting was performed on a BD FACS Aria III appliance. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.