GSM4785609: Patient 9; Homo sapiens; RNA-Seq
CD19+ cells were enriched from the peripheral blood by MACS using StraightFrom Blood CD19 Microbeads. Live CD3- CD14- CD16- CD38high CD27high cells were subsequently sorted by FACS. These were then 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 or 14 cycles of PCR and then used for both 5' gene expression (GEX) and BCR library preparation. BCR target enrichment was achieved using the Chromium Single Cell V(D)J Enrichment Kit for Human B cells. Through fragmentation, adapter ligation and index PCR the final libraries were obtained. The quality of single cell 5' GEX and BCR 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).