GSM7956939: WT Healthy, scRNAseq; Mus musculus; RNA-Seq
Lungs were excised from the chest, washed with PBS, dried on paper towels and then minced into small pieces and suspended in digestion media consisting of RPMI 1640 with 10% FBS, 0.5 ug/mL of DNase I and 1 unit/mL collagenase Type I (Sigma-Aldrich Cat# 0000137295) and placed in an incubator at 37°C for 45 minutes, shaking once during digestion. Lungs were then mashed against 70 μM cell strainers (VWR Cat# 10199-657) to filter and then red blood cells were lysed using ACK lysis buffer, mixed with RPMI 1640 containing 10% FBS and 1% pen-strep, centrifuged at 400g for 5 minutes at 4°C to obtain a single-cell suspension. Cells were then stained and sorted by FACS for alveolar macrophages (CD45+CD11c+SigF+), macrophages (CD45+CD11b+Ly6G-), T cells (CD45+ CD3+) and epithelial cells (CD45-EPCAM+), washed, counted, and then an equal proportion of each population was used for sequencing. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.