GSM4785641: Measles; Homo sapiens; RNA-Seq
Peripheral Blood Mononuclear Cells (PBMCs) were isolated with density gradient seperation and stimulated for 6 hours with anti-CD28 and measles lysate in presence of anti-CD40. Live CD19- CD14- CD3+ CD4+ CD45RO+ CD154+ cells were subsequently sorted by FACS. These were then pooled and applied to the 10X Genomics platform using the Single Cell 5' Library & Gel Bead Kit (10x Genomics) following the manufacturer's instructions. The purified cDNA was amplified using 13 cycles of PCR and then used for both 5' gene expression (GEX) and TCR library preparation. TCR target enrichment was achieved using the Chromium Single Cell V(D)J Enrichment Kit for Human T cells. Through fragmentation, adapter ligation and index PCR the final libraries were obtained. The quality of single cell 5' GEX and TCR libraries was assessed by Qubit quantification and Bioanalyzer fragment analysis (HS DNA Kit, Agilent). The sequencing was performed on a NextSeq500 device (Illumina) using High Output v2 Kits (150 cycles) with the recommended sequencing conditions for 5' GEX libraries (read1: 26nt, read2: 98nt, index1: 8nt, index2: n.a.) and Mid Output v2 Kits (300 cycles) for BCR libraries (read1: 151nt, read2: 151nt, index1: 8nt, index2: n.a., 20% PhiX spike-in).