TCR repertoires of specific V genes, from both adult whole blood and neonatal cord blood samples
TCR mRNA was reverse transcribed off a region towards the 5' end of the constant region. Second strand synthesis was performed using V specific primers directed against V genes TRAV8-4, TRAV21, TRBV12-3 and TRBV20-1. 5' extensions in these primers contained Illumina sequencing primer 2 (SP2) and a random hexamer (6N). Second strand cDNA then underwent 'third strand' synthesis, in which a single strand extension reaction is carried off a relatively nested constant region sequence, in which SP1 and another 6N are introduced. Subsequent PCRs then add the required P5 and P7 elements and amplify up to sufficient concentrations for sequencing (26 cycles total), normalised and sequenced on the Illumina MiSeq. The first six nucleotides of each read relate to the two 6N sequences, which - as they were added before amplification - can be considered molecular barcodes of the original cDNA molecules, and can therefore be used for error-correction purposes. This was performed for both cord blood (CB) and whole blood samples from adult healthy volunteers (HV).