BCR-seq of wild-caught Artibeus jamaicensis: adult male spleen
RNA was extracted from a small piece of splenic tissue by homogenizing the tissue in Trizol and transferring the sample to Qiagen RNeasy columns (Qiagen #74104). 5 RACE ready cDNA was prepared using the SMARTer RACE 5/3 Kit (Takara #634859) using poly-A primers. IGH amplicons were generated via PCR with the 5 RACE Universal Primer and custom constant region primers. Barcodes were added to amplicons from each isotype PCR by amplifying the products with primers using the same 5 RACE Universal Primer and constant region gene-specific regions but with an added barcode and half the Illumina adapter. The second half of the adapter was added in a subsequent PCR step. Barcoded amplicons from different isotypes were pooled and sequenced on an Illumina MiSeq (2 x 300 bp kit).