TCR-Seq of hetereocephalus glaber: adult3 cortical thymus: delta
RNA was isolated from tissues single-cell suspensions with the RNeasy Micro Kit (Qiagen). cDNA was generated performing a template switch anchored RT-PCR. RNA was reverse transcribed via a template switch cDNA reaction using in the same tube 1) a common TRGC primer with degenerate nucleotides specific for all the possible functional TRGC gene segments identified with IMGT annotation rules (5-GAAAGRTATGTYCCAGCCTY-3), 2) a TRDC primer (5-GAGACAAGCAACCTTTGTTCCATT-3) to amplify CDR3sequences, 3) a template-switch adaptor (5-AAGCAGTGGTATCAACGCAGAGTACATrGrGrG-3) and 4) the SuperScript II RT enzyme (Invitrogen). To amplify CDR3and CDR3sequences, we used oligodT primer (5-TTTTTTTTTTTTTTTTTTTTTTTTTVN-3) instead of specific locus primers. The cDNA was then purified using AMPure XP beads (Agencourt). Subsequent amplification of the TRG and TRD region was achieved using a combination of TRGC specific primers including 1) a TRGC2 primer (5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAAAGGGCTTGGGGGAAATGTT-3), 2) a TRGC3 primer (5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCTTGGGGAAAATGTCTGTGTC-3) and 3) a TRGC5 primer (5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGGGGGAAATGTCTCCATCTG-3). These combination of TRG primers was mixed as well with a specific TRDC primer (5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGGTTTGGAAGGAGGCTGAAT-3, adapter in italic) and a primer complementary to the template-switch adapter (5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAAGCAGTGGTATCAACGCAG-3, adapter in italic) with the KAPA Real-Time Library Amplification Kit (Kapa Biosystems). In the case of TRA and TRB sequence the KAPA enzyme and the primer complementary to the template-switch adapter were combined with a TRAC specific primer (5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTTTCAGCTGGTACACGGCA-3) and a TRBC specific primer (5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGATGGCTCAAACACAGCCACC-3). After purification with AMPure XP beads, an index PCR with Illumina sequencing adapters was performed using the Nextera XT Index Kit. This second PCR product was again purified with AMPure XP beads. HTS of the generated amplicon products was performed on an Illumina MiSeq platform using the V2 300 kit, with 300 bp at the 39 end (read 2) and 300 bp at the 59 end (read 1) (at the GIGA center, University of Lige, Lige, Belgium).