GSM9455952: TC, SS3; Mus musculus; RNA-Seq
Single cell suspensions from mLN of 2-week-old (P17) RorcVenus mice were depleted of TCRb, TCRgd, CD19, B220, NK1.1, and Ly6G+ cells via staining with biotinylated antibodies followed by magnetic bead (Dynabeads M-280 streptavidin; Invitrogen) negative selection. Live, Lin(TCRb, TCRgd CD19, B220, NK1.1, Ly6G, SiglecFhi)–CD88–Ly6C–MHCII+RORgt(Venus)+CXCR6– or Lin–CD88–Ly6C–MHCII+RORgt(Venus)–NCAM1+ cells were then sorted into single wells.  Cells were also stained for EpCAM, CD11c, CD11b, CCR6 and NRP1 for acquisition of index sorting information on cell surface expression. Cells were sorted into 3 µL Lysis Master Mix consisting of PEG 8000, Triton X-100, dNTPs, Oligo dT30VN, and RNase Inhibitor in 384-well plates. First-strand cDNA synthesis was done using Maxima H Minus Reverse Transcriptase (ThermoFisher catalog # EP0751) according to the manufacturer's protocol using a custom template-switch oligo in a 2 µM final concentration. cDNA was amplified for 23 cycles using KAPA HiFi HotStart ReadyMix (Kapa Biosystems KK2601) and cleaned up using aMPure XP beads (Beckman Coulter catalog # A63882) at a 0.6X ratio. Full-length cDNA was tagmented using Tn5 (Illumina catalog # 20034198) for 10 minutes at 55°C and libraries were prepared with the Nextera XT DNA Library Preparation Kit (Illumina catalog # FC-131-1024) in a total volume of 6.25 µL with 12 cycles of PCR. Indexed libraries were pooled by volume according to plate quadrant and cleaned by aMPure XP beads (Beckman Coulter catalog # A63882) at a 0.8X ratio. Pools were sequenced on a NovaSeq X in a PE100 run using the NovaSeq X 10B Reagent Kit (Illumina).  An average of 551 thousand paired reads were generated per cell.