GSM8691060: A. fumigatus, Day 7; Mus musculus; RNA-Seq
Sorted pulmonary stromal cells (negative for CD45, CD31 and EpCAM) were collected in a tube containing DMEM (Corning, 10013CV) with 10% FBS at a concentration of 100 cells/μL. The single-cell RNA-Seq assay was performed according to the manufacturer's instructions (Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (Dual Index), 10x Genomics). Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate a gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every GEM was reverse transcribed into cDNA, adding an Illumina TruSeq R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The cell barcoded molecules were then cleaned up with Silane DynaBeads and amplified using 14 PCR cycles.  Next, full-length, barcoded cDNA was then enzymatically fragmented, sized-selected, adapter-ligated, and amplified for library construction. During the library construction, P5, P7, i7 and i5 sample indexes, and TruSeq Read 2 were added. Samples were pooled and run on the NovaSeq 6000 sequencer with a S4 flow cell using the following sequencing parameters: R1: 28 cycles, i7: 10 cycles, i5: 10 cycles, R2: 90 cycles.