design: The adaptor used by the Genome Analyzer II (Illumina, San Diego, CA, USA) was added to the ends of TCR? products by use of 2 sequential rounds of PCR. In the 1st round PCR (PCR-2), 10 ?l of the purified TCR? libraries was amplified with UF primer plus TRBJ_AAA and TRBJ_GAA primers (final 0.05 ?M each) under 30 s at 94°C, 30 s at 50°C, and 90 s at 72°C for 23 cycles, plus a final extension for 10 min at 72°C. PCR products, ranging from 430 to 780 bp, were purified from a 1.5% agarose gel with a Gel Extraction Kit (Qiagen). In the 2nd round PCR (PCR-3), the purified, previous round PCR products were amplified again with A-UF primer and A-TRBJ primers (final 0.1 ?M each), which contain complete adaptor sequences under 30 s at 94°C, 30 s at 65°C, and 90 s at 72°C for 20 cycles, plus a final extension for 10 min at 72°C. Again, the amplified PCR products ranging from 480 to 830 bp were purified the same as the 1st round.
identifiers.namespace: SUB1906195
identifiers.id: t-CS4_CD8_M2