A 5′-RACE [rapid amplification of complementary DNA (cDNA) ends] protocol has been developed for unbiased sequencing of mouse B cell repertoires, as previously described (56). Briefly, RNA (including mRNA) was extracted from total PBMCs of each mouse into 30 μl of water with the RNeasy Mini Kit (Qiagen). 5′-RACE was performed with the SMARTer RACE cDNA Amplification Kit (Clontech). The Ig polymerase chain reactions (PCRs) were set up with Platinum Taq High-Fidelity DNA Polymerase (Life Technologies) in a total volume of 50 μl, with 5 μl of cDNA as template, 1 μl of 5′-RACE primer, and 1 μl of 10 μM reverse primer. The 5′-RACE primer contained a PGM/S5 P1 adaptor, while the reverse primer contained a PGM/S5 A adaptor. We adapted the mouse 3′-Cγ1-3 and 3′-Cμ inner primers as reverse primers for 5′-RACE PCR processing of the heavy chains. A total of 25 cycles of PCR was performed, and the expected PCR products (500 to 600 base pairs) were gel purified (Qiagen). NGS was performed on the Ion S5 system. Briefly, heavy-chain libraries from the same group were quantitated using Qubit 2.0 Fluorometer with the Qubit dsDNA HS Assay Kit and then mixed using a ratio of 1:1:1:1 for sequencing. Template preparation and (Ion 520) chip loading were performed on Ion Chef using the Ion 520/530 Ext Kit, followed by sequencing on the Ion S5 system with default settings. The mouse antibodyomics pipeline was used to process the raw data and to determine the distributions of heavy-chain germline gene usage.

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