design: Reverse transcription (RT) was performed using Clontech SMARTer cDNA template switching: 5' CDS oligo(dT) (12 μM) was added to RNA and incubated at at 72°C for 3 minutes and 4°C for at least 1 minute. The RT mastermix (5x RT Buffer (250 mM Tris-HCl (pH 8.3), 375 mM KCl, 30 mM MgCl2), Dithiothreitol, (DTT 20 mM), dNTP Mix (10 mM), RNAse Out (40U/µL), SMARTer II A Oligo (12 µM), Superscript II RT (200U/µL)) was added to the reaction and incubated at 42°C for 90 minutes and 70°C for 10 minutes. First-strand cDNA was purified using AMPure XP beads (Beckman Coulter catalog# A63882). Following RT, two PCR rounds were carried out to generate immunoglobulin amplicon libraries compatible with Illumina sequencing. The first PCR amplification was carried out using KAPA Real-Time Library Amplification Kit (catalog# KK2702). cDNA was combined with master mix (2X KAPA PCR Master Mix, 12 µM µL 5PIIA and 5 µL IgM/IgG/IgK/IgL Constant Primer (2 µM)). The amplification was monitored using real-time PCR and was stopped during the exponential phase. The amplified products were again purified using AMPure XP beads. A second round of PCR amplification was carried out for addition of barcodes and Illumina adapter sequences: master mix (2X KAPA PCR Master Mix 2x, Nuclease-free water), 10 µM of P5_Seq BC_XX 5PIIA, 10 µM of P7_ i7_XX IgM/IgG/IgK/IgL and were combined with amplified Immunglobulin from the first round PCR and amplified using real-time PCR monitoring. The P5_Seq BC_XX 5PIIA primers contain a randomized stretch of four to eight nucleotides to increase diversity. This was followed by purification with AMPure XP beads. A final PCR step was performed for addition of remaining Illumina adapters by mixing master mix (2X KAPA PCR Master Mix, 10 µM P5_Graft P5_seq, Nuclease-free water), 10 µM of P7_ i7_XX IgM/IgG/IgK/IgL oligo and amplified products from the previous PCR step followed by purification with AMPure XP beads.