GSM8403311: tonsil_GEX_run2; Homo sapiens; RNA-Seq
Single cell suspensions were generated by dissociating tissue (merged from the three technical triplicate wells) using Collagenase IV (Worthington Biochemical) incubated for 30 min at 37°C with gentle shaking as previously described (44). Samples were homogenized with mortar and pestle before filtration over a 100µm cell filter and washed once with cold PBS. Dead cells were depleted from samples using the EasySep™ Dead Cell Removal (Annexin V) Kit (Stemcell Technologies, #17899). Cells were blocked with Human TruStain FcXTM (Biolegend, #422302) as per the manufacturer's protocol. Next, cells from the uninfected tonsil were split into 4 hashing groups (hash #1-4), and cells from the infected tonsil were split into two infected hashing groups (hash #5, 6) and two bystander hashing groups (hash #7, 8). Samples were stained with corresponding hashing antibodies for 30 min at 4°C and washed 3x in FACS buffer (PBS + 1mM EDTA + 2% BSA). Cell suspensions were filtered over a 70µm filter, stained with propidium iodide (PI) for viability, and live/GFP- cells were sorted from the uninfected condition, live/GFP+ cells were sorted from the infected condition, and live/GFP- cells were sorted from the bystander condition on a BD FACS Aria III. Sorted cells were counted and 10,000 cells from the uninfected condition, 5,000 cells from the infected condition, and 5,000 cells from the bystander condition were pooled and processed for scRNA-Seq on the 10x Genomics Chromium platform, utilizing the 10x 3' v3 kit. A scRNA-Seq library was generated as per the manufacturer's protocol and sequenced on an Illumina NextSeq500 instrument. A corresponding library of barcoded hash antibody oligonucleotides was indexed with a standard Illumina D701 index and sequenced as above.