B cell convergence to distinct broadly reactive epitopes following vaccination with chimeric influenza virus hemagglutinins, Guthmiller et al. 2021
This study was designed to analyze the B cell repertoire of cH5/1 binding B cells following vaccination with the cHA platform. Human B cells (CD19+IgM-CD27+cH5/1+) were sorted at day 113 and were used for downstream sequencing. This dataset is comprised of a collection of 5' gene expression datasets from 19 subjects. PROTOCOLS: cH5/1+ memory B cells (CD19+CD27+HA+) were bulk sorted and partitioned into nanoliter-scale Gel Bead-In-Emulsions (GEMs) to achieve single cell resolution using the 10x Genomics Chromium Controller and according to the manufacturer’s instruction (10x Genomics). The sorted single cells were processed according to 5’ gene expression and B cell Immunoglobulin (Ig) enrichment instruction to prepare the libraries for sequencing. Libraries were sequenced using an Illumina HiSeq 4000 at Northwestern University or an Illumina NextSeq 500 at the University of Chicago. Cellranger Single-Cell Software Suite (version 3.0) was used to perform sample de-multiplexing, barcode processing, and single-cell 5’ and V(D)J counting, and Cellranger mkfastq was used to de-multiplex raw base call (BCL) files into sample-specific fastq files. Subsequently, GRCh38-1.2.0 and cellranger-vdj-GRCh38-alta-ensembl-2.0.0 were used as references for the transcriptome and V(D)J assembly, respectively. Cellranger counts and Cellranger vdj package were used to identify gene expression and assemble V(D)J pairs of antibodies. BCR sequences were then processed by IgBlast and our in-house software VGenes.