Systematic Characterization and Comparative Analysis of the Rabbit Immunoglobulin Repertoire
Approximately 0.5 µg of ethanol precipitated RNA was used for first-strand cDNA synthesis according to the manufacturer’s protocol for 5’ RACE using the SMARTer RACE cDNA Amplification kit (Clontech, CA, USA).  The cDNA reaction was diluted into 100 µl of Tris-EDTA buffer and stored at -20°C.  5’ RACE PCR amplification was performed on the first strand cDNA to amplify the VH repertoire with the kit-provided, 5’ primer mix and 3’ rabbit IgG-specific primers RIGHC1 and RIGHC2 (Table S1).  The rabbit VL repertoire was amplified via 5’ RACE, using a 3’ primer mix specific for both the Vκ and Vλ rabbit constant regions.  The VL primers comprised 90% RIGκC mix and 10% RIGλC mix (Table S1) to approximate known ratios of light chain isotypes in rabbits. Reactions were carried out in a 50 µl volume by mixing 35.25 µl H2O, 5 µl 10X Advantage-2 PCR buffer (Clontech), 5 µl 10X Universal Primer A mix (Clontech), 0.75 µl Advantage-2 polymerase mix (Clontech), 2 µl cDNA, 200 nM  VH or VL primer mix, and 200 µM dNTP mix.  PCR conditions were:  95 °C for 5 min, followed by 30 cycles of amplification (95 °C for 30 sec, 60 °C for 30 sec, 72 °C for 2 min), and a final 72 °C extension for 7 min.  The PCR products were gel-purified to isolate the amplified VH or VL DNA (~500 bp). 100 ng of each 5’ RACE amplified VH or VL DNA was processed for Roche GS-FLX 454 DNA sequencing according to the manufacturer’s protocol.  All 454 data were first processed using the sequence quality and signal filters of the 454 Roche pipeline and then subjected to bioinformatics analysis that relied on homologies to conserved framework regions using IMGT/HighV-Quest Tool [22]. Additional filters were applied for full repertoire database construction as follows:  (i) Length cutoff: full-length sequences were filtered by aligned amino acid lengths > 70 residues and aligned framework 4 region lengths > 2 residues; (ii) Stop codons:  aligned amino acid sequences containing stop codons were removed.
UT-Austin
CCH1_VL_RACE_MID4
UT-Austin
HEL1_VL_RACE_RL20