GSM9194505: Biol Rep 1 Tfr; Mus musculus; RNA-Seq
After 3.5 days of cell culture, T cells were harvested, stained, and sorted on a BD FACSAriaFusion cell sorter for FoxP3+CXCR5– (Treg), FoxP3+CXCR5+ (Tfr), and Foxp3–CXCR5+(Tfh) cells among viable CD8–CD19–CD4+ lymphocytes using a 70-micron nozzle. RNA was isolated using TRIzol™ LS Reagent (Invitrogen, #10296028) according to the manufacturer's instructions. RNA was cleaned up with a RNeasy Mini Kit (catalog #74104) according to the manufacturer's instructions. The sequencing library was prepared according to manufacturer´s guidelines with the QuantSeq FWD 3´-mRNA-Seq Kit from Lexogen using 50 ng total RNA as input. Libraries were single-end sequenced with 1x100 bp on a S1 flow cell of an Illumina NovaSeq 6000 instrument to a depth of 10 M raw reads on average at the NGS Core Facility of University Hospital Bonn