scRNAseq_musmusculus_thymus_mouse_CS8D6_female_4week
In vivo Aire mutant:In this project, we performed a fresh thymus enzymatic dissociation (treatment with collagenase plus dispase) from an Aire mutant C57BL/6 mouse heterozygous mutation in Aire exon 6 (del 3554G), which produce a truncated AIRE protein from the AIRE protein SAND domain. Thymic stromal cells together with thymocytes and other cell types present in the thymus gland, showed >90% viability. Cells were processed following the 10X Genomics protocol to prepare a single-cell library using Chromium equipment (10X Genomics). The single cells were isolated and lysed. Next, mRNAs were purified and primed with a poly(T) primer for reverse transcription. Unreactive primers were removed by exonuclease I digestion. Poly(A) tails were added to the first strand cDNA at the 3' end and annealed to poly(T) primers for second-strand cDNA generation. The cDNAs were PCR-amplified, sheared, and prepared into sequencing library. The library was sequenced in an Illumina NovaSeq 6000 sequencer.