Human TRG repertoires of children with CHD and aged-matched controls
mRNA extraction was performed from thawed FACS-sorted cells according to instructions of the RNeasy mini kit (Qiagen). For TRG and TRD repertoire analyses, the isolated mRNA was reverse-transcribed into cDNA using Superscript III enzyme (Invitrogen) and oligo(dT)primers. Amplification of CDR3 regions for TRG clones and TRD clones was performed using a multiplex polymerase chain reaction (PCR). For all conditions and to control PCR contamination, negative controls (H2O) were run together with each PCR, and also subjected to sequencing. All PCR amplicons were subsequently prepared for paired-end Illumina sequencing (v2 500 cycles), using the Illumina MiSeq platform according to the Illumina guidelines.