Profiling Antibody Repertoires in Transgenic Rats

Total RNA from spleens and lymph nodes was extracted (RNeasy Maxi Kit, Qiagen) from each unimmunized heavy chain and kappa chain only transgenic rat (OmniRat, Open Monoclonal Technology Inc., Palo Alto, CA, USA) and antibody sequences were amplified as previously described^{13 (link)} except for different primers used during reverse transcription (Table S2). We chose to interrogate the antibody repertoires found in secondary lymphoid organs as opposed to peripheral blood due to the higher number of B cells. Correct PCR product sizes were verified on an agarose gel (E-Gel EX; Invitrogen) and quantified with fluorometry (Qubit; Life Technologies), pooled at approximately equimolar concentrations and each sample pool was re-quantified before sequencing on an Illumina MiSeq (MiSeq Reagent Kit v3, 600-cycle). All animal experiments were conducted in accordance with the Institutional Animal Care and Use Committee of Scripps Research and approved by the Institutional Research Boards of Scripps Research.