Experimental workflow of repertoire sequencing. (A) Sorted B cells by cell surface markers are prepared for sequencing in bulk or single cell state with further isolation by droplet microfluidics. (B) Templates available for BCR repertoire analysis are both gDNA and cDNA. gDNA contains intron sequences and both V and J genes that are not taken part in V(D)J recombination, resulting in far genomic distance between V region and C region in particular B cell clones. In contrast, only rearranged V(D)J segments exist in cDNA. They are juxtaposed with the C region. (C) For both gDNA and cDNA, a library is constructed by using PCR with multiplex primers targeting multiple V segments. Alternatively, in order to prevent primer bias from a large number of primer sets, a universal forward priming site is attached to the 5′ RACE region by template switching. (D) Once library preparation is completed, NGS platform is chosen considering the depth and length of BCR to be examined.