GSM6797868: Control Tfh_bio rep 1; Mus musculus; RNA-Seq
Enriched CD45.1+&nbsp;GFP OTII CD4 T cells were then stained with CD4, PD-1, PSGL-1, and CXCR5 to sort PSGL-1+CXCR5-&nbsp;non-Tfh cells or PSGL-1loCXCR5+&nbsp;Tfh cells into Trizol LS (Invitrogen). As previously described, cells were prepared for RNA-seq analysis (88, 89). Briefly, total RNA was extracted from the sorted cells (5,000-250,000) using the miRNAeasy micro kit (Qiagen). Approximately 1 ng of isolated RNA was amplified using the Smart-seq2 protocol. Whole transcriptome cDNA was purified using Ampure XP magnetic bead solution (0.8:1 [vol: vol] ratio, Beckman Coulter). Quality control steps were included in determining (i) fragment size distribution quality by capillary DNA electrophoresis, (ii) quantity by fluorescent Picogreen assay (Invitrogen), and (iii) the optimal number of PCR preamplification cycles for optimal cDNA amplification, and (iv) fragment size selection efficiency. S Sequencing was performed according to a paired-end strategy using the NovaSeq sequencer (Illumina). Samples that failed quality control standards were eliminated from further analysis21.