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We separated PBMCs cultured in RPMI-1640 medium (Gibco, CA, USA) supplemented with 10% fetal calf serum (FCS, Gibco, CA, USA), and 200 IU/mL of IL-2 in a 48-well culture plate containing immobilized BCG vaccine at 37°C in 5% CO2. After 72 h, BCG was removed. Cells were cultured until day 12-14. Stored them with RNA protection reagent in -80°C. After reverse transcription into cDNA using Total RNA, the variable region was amplified using cDNA as a template. Then they were sequenced on IIIummia MiSeq. |
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