GSM5294349: Sample_418_RORg_MHCII; Mus musculus; RNA-Seq
RORgt+MHCII+ cells were enriched from a pool of MLN from 3-week-old RorcVenus-CreERT2 mice. Cells were depleted of Lineage (TCRb, TCRgd, CD19, B220, NK1.1)+ cells via staining with biotinylated antibodies followed by magnetic bead negative selection. Live, Lin(CD3, TCRb, TCRgd CD19, B220, NK1.1)–CD64–Ly6C–MHCII+RORgt(Venus)+ cells were then sorted into single wells. Cells were also stained for CD90, CD11c and CD11b for acquiring index sorting information on cell surface expression. Aire+ mTECs were enriched from a pool of thymi from 3-week old mice via staining with biotinylated antibodies against CD45 followed by magnetic bead negative selection. CD45–Epcam+MHCII+Aire(GFP)+ cells were sorted into single wells. Aire+ DCs were enriched from a pool of MLN from the same 3-week old mice. Cells were depleted of Lin+ cells as described above and live, Lin(CD3, TCRb, TCRgd CD19, B220, NK1.1)–CD90–CD64–Ly6C–CD11c+MHCII+Aire(GFP)+ cells were then sorted into single wells. Single cells were sorted into Buffer RLT (Qiagen). Cell lysates were immediately sealed and spun down before transferring to dry ice and storing at -80 ºC. RNA was purified using the Agencourt RNAClean XP beads (Beckman Coulter) at a 2.2X ratio. First-strand cDNA synthesis was achieved using Maxima H Minus Reverse Transcriptase (ThermoFisher) according to the manufacturer's protocol using oligo dT primers, with the addition of a custom template-switch oligo in a 1mM final concentration. cDNA was amplified for 24 cycles using KAPA HiFi HotStart ReadyMix (Kapa Biosystems KK2601). After PicoGreen quantification, 0.1-0.2ng of cDNA was used to prepare libraries with the Nextera XT DNA Library Preparation Kit (Illumina) in a total volume of 6.25µL with 12 cycles of PCR. Indexed libraries were pooled by volume and cleaned by aMPure XP beads (Beckman Coulter) at a 1X ratio. Pools were sequenced on a HiSeq 4000 in a PE50 or PE100 run using the HiSeq 3000/4000 SBS Kit (Illumina).