Bamh1

PfuTurbo DNA Polymerase was obtained from Agilent Technologies (Santa Clara, CA). The restriction enzymes of BamH1 and EcoR1 were purchased from Toyobo Co., Ltd. (Osaka, Japan). The restriction enzymes of Ava1, Sal1 and Xho1 and DNA Ligation Kit were purchased from Takara BIO Inc. (Kyoto, Japan). QIAGEN Plasmid Kits were purchased from QIAGEN, Inc. (Hilden, Germany). INTRON^® A was obtained from Merck & Co., Inc. (Kenilworth, NJ, USA). Mannan was purchased from Nacalai Inc. (Kyoto, Japan). All other chemicals and reagents used were of the highest commercially available quallity, and all solutions were made using deionized and distilled water.

Genomic DNA (3 μg) prepared from MAIT-iPSCs (L7−1 to -6, L11-1 to -4, L15-1 to -3, and L19-1 to -6) with NucleoSpin Tissue (MACHEREY-NAGEL) was digested with 40 U BamH1 (TOYOBO) at 37°C for 18 hr, separated by 0.9% agarose gel electrophoresis, and transferred onto Biodyne membranes (PALL Life Sciences). The membrane was pre-hybridized with PerfectHyb (TOYOBO) at 68°C for 60 min and hybridized with an α-³²P-dCTP-labeled probe at 68°C for 18 hr. The probe was amplified with the primer set TRAV19 (forward) and TRAV19 (reverse) using genomic DNA from C57BL/6 as a template, and the resultant 350 bp PCR product was radiolabeled with the Random Primer DNA Labeling Kit version 2.0 (Takara) (Figure 1—figure supplement 1D). The membrane was washed with 2× saline sodium citrate (SSC) and then 0.1× SSC buffer twice at 68°C, exposed to an imaging plate, and the signal was detected with a phosphoimager (Typhoon 7000, GE Healthcare).