GSM9207310: mTreg_cKO; Mus musculus; RNA-Seq
Regulatory T cells (Tregs) were isolated from Srsf3fl/+ Foxp3Cre-YFP female mice spleen by MACS using CD4+CD25+ Regulatory T Cell Isolation Kit according to the manufacturer's instructions. Single-cell cDNA was generated using the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (Dual Index, 10× Genomics), amplified by PCR, and purified with SPRIselect magnetic beads (Beckman Coulter) according to the protocol. Full-length cDNA was enriched via biotin–streptavidin capture with Dynabeads M280 Streptavidin, then processed using the Oxford Nanopore Ligation Sequencing Kit V14 (SQK-NBD114.96) following manufacturer's guidelines. After barcoding with the Native Barcode Kit (NB01–NB96, SQK-NBD114.96)  and a 1× AMPure XP bead cleanup, 300 fmol of adapter ligated library was loaded onto a PromethION flow cell (vR10.4.4). The PromethION platform (Oxford Nanopore Technologies, ONT) generates an expression matrix with both gene-level (total gene expression) and transcript-level (individual isoform counts) data when used for single-cell third-generation sequencing, thanks to its full-length mRNA sequencing advantage.