Chromium next gem single cell 3 gel beads v3.1 kit

Cells were washed and trypsinized using ReagentPack™ Subculture Reagents (Lonza Bioscience). Single cell suspensions were washed with PBS + 5% FBS, resuspended in 200 μL PBS + 5% FBS, passed through a 70 μm filter, and placed on ice. Cells were counted using trypan blue exclusion on a Luna II (Logos Biosystems). The appropriate numbers of cells to achieve a targeted cell input of 5,000 cells per condition were used to generate the GEMs (Gel Bead-In Emulsion). The Chromium Next GEM Single Cell 3’ Gel beads v3.1 kit (10X Genomics, Pleasanton, CA, USA) was used to create GEMs following manufacturer’s instruction. All samples and reagents were prepared and loaded into the chip and ran in the Chromium Controller for GEM generation and barcoding. GEMs generated were used for cDNA synthesis and library preparation using the Chromium Single Cell 3’ Library Kit v3.1 (10X Genomics) following the manufacturer’s instruction.

scRNAseq data of ALI cultures were obtained from Ravindra et al.¹² (link) Briefly, human bronchial epithelial cells (HBECs, Lonza) were cultured at an air-liquid interface (ALI) for 28 days to achieve full mucociliary differentiation. Cultures were then challenged at the apical surface with 10⁴ plaque forming units (PFU) of SARS-CoV-2. An uninfected mock control and samples at 1dpi, 2dpi, and 3dpi were harvested with TrypLE Express Enzyme (ThermoFisher) and prepared with the Chromium Next GEM Single Cell 3’ Gel beads v3.1 kit (10X Genomics), at a target loading of 10,000 cells per sample. Libraries were generated using the Chromium Single Cell 3’ Library Kit v3.1 (10X Genomics) and sequenced on the NovaSeq 6000 using HiSeq 100 base pair reads and dual indexing, to an average depth of 31,383 reads per cell. Data were aligned and pre-processed using the 10x Genomics Cell Ranger pipeline and a combined human and SARS-CoV-2 genome. On average across samples, there were 10,000 to 15,000 counts per cell and 2,400 to 3,600 unique genes per sample. For more information, see original publication.¹² (link)