GSM4785642: Patient 1; Homo sapiens; RNA-Seq
CD45+ cells were enriched from bronchoalveolar lavage samples by MACS using CD45 Microbeads. Live CD45 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 cycles of PCR and then used for both 5' gene expression (GEX), TCR and BCR library preparation. TCR and BCR target enrichment was achieved using the Chromium Single Cell V(D)J Enrichment Kit for Human T cells and Enrichment Kit for Human B cells, respectively. Through fragmentation, adapter ligation and index PCR the final libraries were obtained. The quality of single cell 5' GEX, TCR 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 TCR and BCR libraries (read1: 151nt, read2: 151nt, index1: 8nt, index2: n.a., 20% PhiX spike-in).