Illumina next-generation sequencing (NGS)

Total RNAs of splenocytes collected from naive and immunized gl-CH31 dKI mice were extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. Reverse transcription was performed using SuperScript IV with random primers, also according to the manufacturer instructions. After cDNA synthesis, knocked-in gl-CH31 V_HDJ_H rearrangements were amplified via 1st Round PCR using P5-H10-leader paired to P7 plus IgM, IgG1, IgG2b, IgG2c IgG3 or mouse IgA reverse primers. Likewise, knocked-in VJ rearrangements were amplified by PCR using P5-ox-leader primers paired to the P7-mouse kappa reverse primer. Phusion Hot Start Flex DNA polymerase (NEB, Cat# M0535) was used as polymerase enzyme. PCR products were gel-purified using QIAquick Gel extraction Kit (Qiagen) and Bar codes and Illumina sequencing tags were added to the purified amplicons by 2nd round PCR using Index Kit barcode-tagging primers (Illumina). The bar-coded PCR amplicons were individually purified with QIAquick Gel Extraction Kit (Qiagen) and quantitated by qPCR (Kapa Sybr fast qPCR kit, Kapa Biosystems). The purified individually bar-coded amplicons were then pooled together at equal molar DNA. Pooled amplicons were further quantitated by qPCR, diluted at 4 nM, denatured and mixed with the denatured PhiX, and finally, loaded onto Illumina MiSeq kit V3 (2 × 300 base pairs; Illumina) cartridges for deep sequencing on an Illumina NovaSeq 6000 sequencer at the UCSD genomics core. After NGS, two mice in the “SOSIP long” group were determined to have genotypes inconsistent with the intended gl-CH31 dKI (heterozygous double knock-in; V_HDJ_H^+/- x V_κJ_κ^+/-) genotype and were excluded from the study. Two additional mice were determined to have NGS libraries contaminated with non-gl-CH31 reads and were also excluded from the study.