| Construction Protocol | RNA was extracted using Isogen protocol and cDNA synthesized using the
SuperScript II kit (Invitrogen) with oligo-dT primers (Invitrogen)
according to manufacturer?s protocol. cDNAs were then amplified using
ExTaq polymerase kit (TAKARA). TRAV14 (CCAATCTCTGACAGTCTGGGAA) and TRAC
(AACTGGTACACAGCAGGTTCTG) primers were used for TRAV14 repertoire; TRBV2
(CGAACAGTATCTAGGCCACAATGC) and TRBC (GCACACGAGGGTAGCCTTTTGTTT) primers
for TRBV2 repertoire. PCR program was as follow: 4 min at 94 ?C; x
cycles of 30 s at 94 ?C, 30 s at 62 ?C, 30 s at 72 ?C; 5 min at 72 ?C 5;
hold at 4 ?C. The number of cycles was adapted to the initial number of
cells (24 or 25 cycles for conventional T cells, 27 to 29 for regulatory
T cells) as we observed the formation of heteroduplexes in saturated
reactions. PCR products were sequenced on a Roche Genome Sequencer FLX
Titanium system after adapter ligation following standard procedures. |
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