Unbiased BCR Repertoire Analysis

BCR repertoire analysis was performed using unbiased next-generation sequencing developed by Repertoire Genesis, Inc. (Osaka, Japan) [11 (link)]. Briefly, cDNA was synthesized from total RNA using the polyT18 primer (BSL-18E) and Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA). After synthesizing double-strand (ds)-cDNA, the P10EA/P20EA dsDNA adaptor was ligated and cut with the NotI restriction enzyme. Nested PCR was performed with KAPA HiFi DNA Polymerase (Kapa Biosystems, Woburn, MA, USA) using IgG constant region-specific primers (CG1 and CG2) and P20EA. The amplicon library was prepared by amplification of the second PCR products using P22EA-ST1 and CG-ST1-R. Index (barcode) sequences were added by amplification with the Nextera XT Index Kit v2 Set A (Illumina, San Diego, CA, USA). Sequencing was performed using the Illumina MiSeq paired-end platform (2 × 300 bp). BCR sequences were assigned based on identity with reference sequences from the international ImMunoGeneTics information system® (IMGT) database (http://www.imgt.org) using repertoire analysis software originally developed by Repertoire Genesis, Inc. (Osaka, Japan).