GSM7774994: FoxP3 cells, split into FoxP3+LAP- and FoxP3+LAP+; Mus musculus; RNA-Seq
Spleens were harvested and CD4+ T cells magnetically isolated. Cells were stained with CD25 (FoxP3) and LAP antibodies and sorted into FoxP3-LAP+ or FoxP3+ populations using FACS Aria II. Approximately 10,000 targeted cell recovery per sample was applied to a single cell master mix with lysis buffer and reverse transcription reagents, following the Chromium NextGEM Single Cell 3' Reagent Kits User Guide, CG000204 Rev D (10X Genomics, Inc, Pleasanton, CA). Along with the single cell gel beads and partitioning oil, the single cell master mixture containing the single cell suspension was dispensed onto a Single Cell Chip G in separate wells, and the chip loaded to the Chromium Controller for GEM generation and barcoding, followed by cDNA synthesis and library preparation. At each step, the quality of cDNA and library was examined by Bioanalyzer and Qubit. The resulting barcoded libraries were pooled in equal molarity and sequenced in a custom program for 28bp plus 91bp paired-end sequencing on Illumina NovaSeq 6000 (Illumina, Inc). Approximately 50K reads per cell were generated.