Nucleospin extract 2 gel extraction kit

Antigen-specific CD4⁺ T cell clones were stained as described above for viability and surface expression of CD3 and CD4 (reagent details in Key Resources Table). A maximum of 5 × 10³ live CD3⁺CD4⁺ T cells was sorted into 100 μL of RNAlater (Ambion) using a FACSAria III (BD Biosciences). Sorted cells were stored at −80°C. All expressed TRA and TRB gene transcripts were amplified using an unbiased template-switch anchored RT-PCR (Quigley et al., 2011 ). Pure mRNA was isolated from sorted cells using μMACS Oligo(dT) MicroBeads (Miltenyi Biotec), and cDNA was generated using a SmarterRACE Kit (Takara) with Superscript II (Invitrogen). Transcripts were amplified using an Advantage 2 PCR Kit (Takara) with the relevant constant region primers (reagent details in Key Resources Table). Amplicons were separated on agarose gels and extracted using a NucleoSpin Extract II Gel Extraction Kit (Macherey-Nagel). Subclones were generated using a TOPO TA Cloning Kit (Invitrogen) and One Shot Max Efficiency DH5α-T1 Competent Cells (Invitrogen). Conventional sequencing was performed as described previously (Price et al., 2005 (link)). Gene use was assigned using the ImMunoGeneTics (IMGT) nomenclature (Lefranc, 2003 (link)).