Profiling B cell immunodominance after SARS-CoV-2 infection reveals antibody evolution to non-neutralizing viral targets, Dugan et al. 2021
Description
This study was designed to capture the single cell transcriptome and BCR sequence of B cells that were bait sorted using oligo-tagged viral antigens. Human B cells (viable/CD19+/antigen-PE+ or viable/CD19+/antigen-APC+) were bait sorted after initial magnetic enrichment and used in downstream sequencing. PROTOCOLS: PBMCs were isolated from whole blood (acute samples) or leukoreduction filters (convalescent samples) by use of a Lymphoprep Ficoll gradient and any contaminating red blood cells were lysed by ACK buffer. Cells were then frozen at -80 deg C in fetal bovine serum supplemented with 10% DMSO. On the day of sorting and sequencing cells were thawed and B cell were purified by human pan B cell EasySep enrichment kit from STEMCELL technologies. Cells were partitioned into nanoliter-scale Gel Bead-In-EMulsions (GEMs) to achieve single cell resolution for a maximum of 10,000 individual cells per sample. Utilizing the Chromium Single Cell A Chip Kit(10x Genomics, PN-120236) and Chromium Single Cell 5' Library & Gel Bead Kit (10x Genomics, PN-1000006) , poly-adenylated mRNA from an individual cell was tagged with a unique 16 base pair 10x barcode and 10 base pair Unique Molecular Identifier. Libraries were generated using the Chromium Single Cell 5' Feature Barcode Library Kit (10x genomics, 1000080) and the Chromium Single Cell V(D)J Enrichment Kit Human B Cell (10x Genomics, PN-1000016). The cDNA amplicon size for each library was optimized using enzymatic fragmentation and size selection and the concentration and average fragment size of each library was determined using an Agilent High Sensitivity DNA Kit (Agilent). Specifically, user guide CG000186 Rev D was used for all steps. All libraries were sequenced on an Illuina NextSeq550 or an Illumina NextSeq500 with 26 cycles apportioned for read 1, 8 cycles for the i7 index, and 134 cycles for read 2 using an NextSeq 500/550 High Output Kit v2.5 (150 Cycles). Some samples were sequenced using a NextSeq 550 housed within the Pritzker School of Molecular Engineering's Chicago Immunoengineering Innovation Center (Chicago, IL) and others on a NextSeq500 belonging to the University of Chicago Genomics Facility (Chicago, IL - RRID: SCR_019196) DATA PROCESSING PIPELINE: Demultiplexing the Illumina sequencer's base call files (BCLs) for each flowcell directory into FASTQ files was performed by "cellranger mkfastq" (cellranger version 3.02) Gene counts were calculated using "cellranger count" (cellranger version 3.02) under default setting， BCR sequences were assembled using "cellranger vdj" (cellranger version 3.02) under default setting. GRCh38-1.2.0 for transccriptome, cellranger-vdj-GRCh38-alts-ensembl-2.0.0 for V(D)J