GSM9107861: C038_1_HTO; Homo sapiens; RNA-Seq
The cryopreserved mononuclear cell suspensions from the tonsil samples were thawed and processed to form single-cell suspensions. The samples were stained for surface antigen expression via the indicated antibodies and TotalSeq HashTags (BioLegend) for 30 min at 4°C, followed by viability dye staining for 15 min at 4°C. Sample suspensions were sorted via a BD FACSAria Fusion Flow Cytometer (BD Biosciences) to isolate CD3+CD4+CD45RA-CD45RO+CXCR5+CD27+ TFH cells. Equal numbers of sorted cells from each tonsil sample were pooled and stained with TotalSeq-A Human Universal Cocktail in accordance with the manufacturer's protocols. Sequence tags of DNA-barcoded mAbs for CITE-seq were incorporated into existing scRNA-seq methods to enable simultaneous quantification of protein and gene expression by single cells. A total of 40,000 cells from the stained TFH pool were subjected to 3'-tag capture via the 10X Genomics Chromium Controller. Library preparation was conducted for sequencing according to the manufacturer's protocols. Next-generation sequencing was performed using the Illumina NovaSeq 6000 System.