Total RNA was extracted from rs2013278 allele knock-in clones using an RNeasy kit (QIAGEN, Valencia CA). Next, we synthesized first-strand cDNAs using a High-Capacity cDNA Reverse Transcription kit (Thermo-Fisher Scientific). RT-PCR was performed using the primers shown in Additional file 5 and Ex Taq polymerase (Takara-bio, Kusatsu, Japan). Preliminary experiments showed that 33 cycles were optimal for achieving linear amplification of each CD28 splicing isoform. Quantitation of each transcript was performed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Differences in expression levels of CD28 isoforms in cells of the two rs2013278 genotypes (i.e., AA and TT) were analyzed using the Mann–Whitney U test. These experiments were repeated 3 times with essentially identical results.
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