M mlv reverse transcriptase and oligo dt

CD5^lo, Ly6C⁻, and Ly6C⁺ naive CD8⁺ T cells were purified from pulled spleens and LNs (or from thymus). RNA was isolated with NucleoZOL (Macherey-Nagel) and cDNA was synthesized with M-MLV reverse transcriptase and oligo dT (TAKARA). Real-time RT-PCR was performed with the TaqMan Gene Expression Master Mix using StepOnePlus Real-Time PCR System (Applied Biosystems) using StepOne Software (Applied Biosystems). Detailed information of the primers used is described in Supplementary Table 3.

CD44^loCD5^loLy6C⁻, CD44^loCD5^hiLy6C⁻, and CD44^loCD5^hiLy6C⁺ CD8⁺ T_N cells were sorted from pulled spleens and LNs. RNA was isolated with NuleoZOL (Macherey-Nagel) and extracted RNA was measured by NanoDrop and 1 μg of RNA was reverse transcribed into cDNA with M-MLV reverse transcriptase and oligo dT (TAKARA), following manufacturer’s instructions. Real-time RT-PCR was performed with the TaqMan Gene Expression Master Mix using StepOnePlus Real-Time PCR System version 2.2.2 (Applied Biosystems). The following TaqMan probes (Applied Biosystems) were used: Rorc (Mm01261022), Il17a (Mm00439618), Irf4 (Mm0516431), Ifnγ (Mm01168134), Tbx21 (Mm0045960), Eomes (Mm01351984), and Rn18sRn45s (Mm03928990). The expression level was calculated as 2-DCT, where DCT is the difference between the CT values of the target gene and 18S.

1–2 × 10⁶ spleen cells or FACS-purified CD4⁺ T cells from the indicated mice were used for RNA extraction with NucleoZOL (Macherey-Nagel) and stored at -80 °C before the further steps. Isolation of mRNA was done according to the manufacturer’s instructions. cDNA was synthesized with the M-MLV reverse transcriptase and oligo dT (TAKARA). Real-time RT-PCR was performed with the TaqMan Gene Expression Master Mix using StepOnePlus Real-Time PCR System with TaqMan probe for Il2 mRNA (Mm00434256_m1; Applied Biosystems).