Single-cell suspensions from BM of 3x NP-CGG immunized C57BL/6 mice were prepared and CD19+ cells were enriched by magnetic cell sorting using anti-CD19 microbeads (Miltenyi Biotech). Ex vivo IgG1+/IgG2b+CD19+CD38+GL7-CD138-IgM-IgD- memory B cells were isolated by FACS (Influx cell sorter (BD Bioscience)) and applied to the 10X Genomics platform using the Single Cell 5' Library & Gel Bead Kit (10x Genomics) following the manufacturer's instructions. The amplified cDNA was used for simultaneous 5' gene expression (GEX) and murine BCR library preparation. BCR transcripts were additionally amplified by Chromium Single Cell V(D)J Enrichment Kit for murine B cells (10x Genomics). Upon adapter ligation and index PCR, the quality of the obtained cDNA library was assessed by Qubit quantification, Bioanalyzer fragment analysis (HS DNA Kit, Agilent) and KAPA library quantification qPCR (Roche). The sequencing was performed on a NextSeq500 device (Illumina) using a High Output v2 Kit (150 cycles) with the recommended sequencing conditions (read1: 26nt, read2: 98nt, index1: 8 nt, index2: n.a.) for the transcriptome and Mid Output v2 Kit (300 cycles) for BCR repertoire analysis (read1: 150nt, read2: 150nt, index1: 8nt, index2: n.a., 20% PhiX spike-in).
GEO Accession
GSM4147196