03-2017sort
First, one-step RT-PCR was performed with the following primers: an alpha constant region primer, a beta constant region primer, and a mixture of 23 alpha and 19 beta variable region primers. Next, a PCR was performed to amplify more internally, again with constant region primers and a mixture of variable region primers. Finally, a third PCR was performed in two separate plates, one for alpha chains and one for beta chains, to add row/plate barcodes as well as column/chain barcodes. Alpha and beta chain PCR products were separately pooled and run on gels to allow exclusion of primer dimers from extracted DNA. After gel extraction, samples were sequenced at the MIT BioMicroCenter using Illumina MiSeq 500nt paired-end kit.