GSM5058861: CMV_CD4_pd1_pos_neg_96_well_TCR; Homo sapiens; OTHER
Class-II tetramer+ cells were sorted on a BD FACSMelody cell sorter into 96 well PCR plates containing 10ul/well of a 10mM Tris and 500U/ml RNasin Inhibitor solution. TCR sequencing was performed as previously described (Han et al., 2014). Briefly, nested PCR of the TCR alpha and beta CDR3 region was performed by firstly using a Onestep RT-PCR kit (Qiagen) for cDNA synthesis and first round PCR, followed by second amplification with HotStar Taq (Qiagen). Finally a barcoding reaction was carried out using HotStar Taq. PCR products were then pooled and gel purified (Qiagen) paired end sequencing reaction using a MiSeq v2 500 sequencing kit (Illumina)