Transcriptional dissection of the murine thymus gland at single-cell resolution

Transcriptional dissection of the murine thymus gland at single-cell resolutionIn vitro Aire mutant mTECs :In this project, we co-culture murine Aire mutant mTEC cells together with thymocytes (mTEC 3.10 cell line harboring a heterozygous mutation in Aire exon 6 (del 3554G), which produce a truncated AIRE protein at the Aire exon 6 that encodes the AIRE protein SAND domain. Mutant mTEC cells plus fresh SP TCD4+ thymocytes were co-cultured for 36 hours. Co-culture showed >90% viability. Cells were processed following the 10X Genomics protocol to prepare a single-cell library using Chromium equipment (10X Genomics). Next we used the Illumina protocol: The single cells were isolated and lysed, the 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.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.