Unbiased B-cell Library Preparation for MiSeq

B-cell libraries for Illumina MiSeq sequencing were prepared, as follows. Total RNA was extracted from 5 to 10 million PBMCs using the AllPrep DNA/RNA Mini Kit (Qiagen). In an effort to generate unbiased B-cell libraries, cDNA synthesis was subsequently performed using the Takara Clontech SMARTer RACE cDNA Amplification Kit using primers with specificity to IgG, IgM, IgK and IgL. The subsequent RACE-ready cDNA was diluted in Tricine-EDTA according to the manufacturer's recommended protocol. First-round Ig-encoding sequence amplification was performed using AccuPrime Pfx Supermix (Invitrogen, Waltham, MA, USA), containing gene-specific primers (120 nm) and 1 × concentration of Takara/Clontech 10 × Universal primer mix. Amplicons were purified using FlashGels (Lonza, Allendale, NJ, USA) and used as templates for second-round PCR amplification. A second-round PCR amplification (10 cycles) was performed in order to add MiSeq adapter sequences to both ends of the amplicon. After re-purification, a final 5-cycle amplification was performed by adding P5 and P7 index sequences for Illumina sequencing. Purified, indexed libraries were quantitated using the KAPA library quantification kit (Kapa Biosystems, Wilmington, MA, USA) performed on an Applied Biosystems 7500 Fast real-time PCR machine (Applied Biosystems, Foster City, CA, USA).