GSM7780306: Thymic stromal cells, IRF6flox, scRNAseq; Mus musculus; RNA-Seq
Single cell suspensions were generated from target tissues from specific genotype mice. For venus-positive or venus-negative cell populations, cells were sorted by FACs into collection tubes. Cell suspensions were stored on ice, counted and viabilities checked prior to be loaded on the 10x Genomics chromium platform for single cell partitioning, lysis, and reverse transciption of mRNA into cDNA libraries. Library constructionwas performed according to the manufacter's instructions (Single Cell 3' v3 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 retrotranscribed to cDNA, which contains a partial 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 fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.