GSM5494129: CTX_CD4_rep5; Mus musculus; OTHER
On day 25, the lymph nodes and spleen were gently disrupted and filtered through a 40 μm nylon cell strainer to make single-cell suspensions. Cells were treated with RBC lysis buffer before use. After staining with anti-CD8 and anti-Thy1.1 fluorochrome-conjugated antibodies, Thy1.1-negative CD8-positive endogenous T cells were isolated by fluorescence-activated cell sorting. Total RNA of each sample was extracted using the RNeasy Plus mini kit (Qiagen). To generate the TCR sequencing library, 100 ng RNA per sample was reverse transcribed and amplified using nested polymerase chain reaction with a SMARTer mouse TCR a/b profiling kit (Takara Bio). Illumina-specific adaptor sequences were incorporated during amplification. The TCR sequencing library was size-selected and purified using AMPure XP beads (Beckman Coulter).