Quantitative (q)-RT-PCR assays were performed as described (6 (link)) except that primers for 18S rRNA were used (10 (link)) to normalize samples. Quantitative circle transcript (CT) RT-PCR
assays were carried out as described (11 (link)) with
modifications. Primers γ2bF and γ3R were used for Iγ2b-Cγ3
CT assays (Table 1 ). Iγ2b-Cμ CTs
were detected using the Iγ2bF and CμR.1 primers (12 (link)) (Table 1 ). Semi-quantitative
RT-PCR for Iε−Cμ CTs was performed using primers IεF and
CμR with Phire Hot Start II polymerase (Thermo Scientific) and an initial
denaturation for 5 min at 98°C followed by 34 cycles of, 98°C for 5 sec,
60°C for 5 sec, 72°C for 8 sec, both in a 25 ul reaction on 5 fold serial
diluted cDNA. 3D DNA FISH was carried out as described (13 (link)).
assays were carried out as described (11 (link)) with
modifications. Primers γ2bF and γ3R were used for Iγ2b-Cγ3
CT assays (
were detected using the Iγ2bF and CμR.1 primers (12 (link)) (
RT-PCR for Iε−Cμ CTs was performed using primers IεF and
CμR with Phire Hot Start II polymerase (Thermo Scientific) and an initial
denaturation for 5 min at 98°C followed by 34 cycles of, 98°C for 5 sec,
60°C for 5 sec, 72°C for 8 sec, both in a 25 ul reaction on 5 fold serial
diluted cDNA. 3D DNA FISH was carried out as described (13 (link)).
