GSM5694066: Splenic YFP+ Tregs, WT; Mus musculus; RNA-Seq
Spleen tissues were processed with the flat end of a syringe in a 100 mm culture dish containing 5 ml cold FACS buffer (2% FBS in PBS), then passed through a 70 μm cell strainer into a 15 ml tube. Cells were centrifuged to remove the supernatant. Cell pellets were treated with 1ml ACK (Ammonium-Chloride-Potassium) Lysing Buffer to remove the red blood cells. After washing with 10 ml cold FACS buffer, the remaining cells were stained with 7AAD (Part 76332; Lot B226294 Biolegend) for 30 min at 4 °C, then washed and resuspended in cold FACS buffer for flow cytometric sorting using FACS Aria II Cell Sorter (BD Biosciences). Sorted YFP+7AAD- cells with a viability higher than 90% were used for 10X genomics scRNA-seq. Finally, libraries were sequenced on a NovaSeq 6000 system with NovaSeq 6000 S4 Reagent Kit (300 cycles). RNA libraries were prepared for sequencing using standard Illumina protocols