Single-cell Profiling of Human Airway Epithelium

HAE cultures maintained for 3 weeks post-airlift were processed for scRNA-Seq. Single cell suspensions of HAE cultures were prepared as described previously^{27 (link)}. To enable multiplexing and doublet detection, cells were labeled with barcoded antibodies described previously^{28 (link)}. Briefly, approximately 200,000 cells per sample were resuspended in staining buffer (PBS, 2% BSA, and 0.01% Tween) and incubated for 10 minutes with Fc block (TruStain FcX (BioLegend) and FcR blocking reagent (Miltenyi). Cells were then incubated with oligonucleotide-conjugated hashing antibodies (generated in-house by the New York Genome Center as described²⁹ ) for 30 min at 4°C. After labelling, cells were washed 3 times in staining buffer. After the final wash, cells were resuspended in PBS plus 0.04% BSA, filtered, and counted. Cells were pooled (6 samples per pool), super-loaded to the 10X Genomics Chromium Controller (~4,170 cells per sample, ~25,000 cells per lane) and processed with the Chromium Next GEM single-cell 5′ library and gel bead kit according to manufacturer’s protocols. Hashtag-oligonucleotide (HTO) additive oligonucleotide primer was spiked into the cDNA amplification PCR, and the HTO library was prepared as described previously²⁹ .