Barcoding Protocol for Single-Cell Analysis

Barcoding of individual biological samples was performed as described by McGinnis et al., 2019b (link). In summary, dissociated lung cells (0.5 × 10⁶ cells per sample) were suspended in 150 μLs solution containing a 1:1 molar ratio (200 nM) of anchor and barcode oligonucleotide containing a unique sequence for each of the 12 samples to be processed. Samples were incubated for 13 min at room temperature with gentle mixing every 3–5 min. Next, a co-anchor (200 nM) was added to stabilize barcodes within the membrane and incubated for additional 5 min. Cells were washed twice in PBS and cell counts were measured using a Countess automated counter (Thermo Fischer Scientific, Burlington, ON, Canada) and viability was measured based on the ratio of cells staining with trypan blue (Thermo Fischer Scientific, Burlington, ON, Canada). Equal ratio of cells from each 12 barcodes was pooled and 1000 cells/μL were further processed through the 10x-Genomics pipeline. Only samples with viability >85% were used.