GSM9458105: TC_MLN_RANK, GEX; Mus musculus; RNA-Seq
RORgt+CXCR6–MHCII+ cells were FACS-isolated from mLN of 2-week-old (P16) RorcVenusTnfrsf11afl/fl (n = 4 biological replicates) and RorcVenusRorgtcreTnfrsf11afl/fl mice (n = 6 biological replicates). Lymph nodes were processed as outlined above. Each biological replicate single cell suspension was hashtagged with BioLegend TotalSeq-B Hashtag antibodies. Live, Lin(TCRb, TCRgd, CD19, NK1.1, SiglecFhi)–CD64–Ly6C–CD90.2+MHCII+RORgt(Venus)+CXCR6– cells were then sort purified. Cells were sorted into cRPMI-20% FBS before being pelleted and resuspended in cRPMI-2% FBS. scRNA-Seq of FACS-sorted cell suspensions was performed on Chromium instrument (10X Genomics) following the user guide manual for 3′ v3.1. In brief, cells were washed once with PBS containing 1% bovine serum albumin (BSA) and resuspended in PBS containing 2% BSA to a final concentration of 700–1,300 cells per ml. Cell viability was confirmed to be >80% by 0.2% (w/v) Trypan Blue staining (Countess II). Cells were captured in droplets. Following reverse transcription and cell barcoding in droplets, emulsions were broken, and cDNA was purified using Dynabeads MyOne SILANE followed by PCR amplification as per manufacturer's instructions. ~10,000 cells were targeted. Samples were multiplexed on one lane of 10X Chromium using HashTag Oligonucleotides (HTO) following a previously published protocol88. Libraries were sequenced on an Illumina Novaseq X with standard dual indexing and demultiplexing.