title: GSM5365669: Group4_GEX; Homo sapiens; RNA-Seq
library_construction_protocol: Cells were enriched from the peripheral blood by MACS using StraightFrom Blood CD3, CD19 and CD138 Microbeads. Cells were subsequently incubated with barcoded and fluorochorme-conjugated antibodies and sorted by FACS as live CD3+CD14-CD16-CD38+HLA-DR+, live CD3-CD14-CD16-CD38lowCD27+ and live CD3-CD14-CD16-CD38highCD27high. The barcoded antibodies used for cell-surface protein analysis were anti-human CD138 (GTATAGACCAAAGCC), CD20 (TTCTGGGTCCCTAGA), CD4 (GAGGTTAGTGATGGA), CD8 (GCGCAACTTGATGAT), CD45RO (CTCCGAATCATGTTG), CD45RA (TCAATCCTTCCGCTT), CD25 (TTTGTCCTGTACGCC), CD127 (GTGTGTTGTCCTATG) and CD154 (GCTAGATAGATGCAA). Sorted populations from three non-reponding and one reponding donor were pooled in equal proportions 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 12 cycles of PCR and was subsequently used for 5' gene expression (GEX), cell-surface protein, TCR and BCR library preparation acording to 10x Genomics Chromium Next Gem Single Cell V(D)J Reagent Kits v1.1 with Feature Barcoding technology for Cell Surface Protein. The quality of the libraries was assessed by Qubit quantification and Bioanalyzer fragment analysis (Hight Sensitivity DNA Kit, Agilent). The sequencing was performed on a NextSeq2000 device (Illumina) using P3 reagent kit (200 cycles) with the recommended sequencing conditions for 5' GEX and cell-surface protein libraries (read1: 26nt, read2: 98nt, index1: 8nt, index2: n.a.) and P3 reagent kit (300 cycles) for TCR and BCR libraries (read1: 151nt, read2: 151nt, index1: 8nt, index2: n.a., 2% PhiX spike-in).