GSM8750823: Cre negative, H2-Ab1-flox/flox replicate 2; Mus musculus; RNA-Seq
At 3 weeks post-infection, lungs were harvested and single cell suspensions were generated by mechanical homogenization at 4ºC without tissue digestion. Cells for encapsulation were enriched by cell sorting to exclude cells expressing the following markers: CD90, B220, CD19, NK1.1, Ly-6G, Siglec-F. IMDM complete media (Gibco IMDM supplemented with 10% fetal bovine serum, 200 µM beta-mercaptoethanol, and Gibco supplements Glutamax, pyruvate, non-essential amino acids) was used to prepare sorted cells. Approximately 30,000 cells were sorted for each sample. The cells were washed twice in PBS by centrifugation prior to encpasulation. Library construction was performed according to the manufacturer's instructions (10X Genomics Chromium Next GEM Single Cell 3' Gene Expression Kit v.3.1 (dual index)). Cells in samples 1 through 4 were encapsulated in separate lanes of the same 10X chip. Encapsulation and library preparation was performed on each sample side-by side rather than pooling samples. Briefly, gel bead-in-emulsion (GEMs) were generated on the 10X Chromium X controller. Barcoded cDNA was generated using reverse transcription and barcoded adaptor oligonucleotides. The cDNA was amplified by PCR, then enzymatically fragmented to optimize cDNA amplicon size for sequencing. P5, P7, i7 and i5 sample indexes and TruSeq read 2 sequences were added via end repair, A-tailing, adaptor ligation and PCR.