Mj opticon 2 thermo cycler

Total mRNA from 3–5 × 10⁶ CD4⁺ T cells after overnight anti-CD3 and anti-CD28 stimulation was isolated using RNAeasy Mini Kit (Qiagen) and quality and quantity were verified using a Nanodrop (Thermo scientific). cDNA synthesis was carried out using SuperScript III First-strand Synthesis SuperMix for qRT-PCR (Invitrogen). Real-time PCR was carried out using the MJ Opticon 2 thermo cycler (MJ Research) and Taqman probes (GATA3, NFIL3, c-Maf, IL-10, HPRT1; Life Technologies). Expression of the target gene was obtained by using the following equation: target gene expression = 2^{(mean HPRT1 ct − mean target gene ct)}.

ChIP was carried out using the EZ-Magna ChIP A Chromatin Immunoprecipitation Kit (Millipore). A total of 20 × 10⁶ CD4⁺ T cells were fixed in 1% formaldehyde (Fisher Scientific). Glycine buffer was added to quench any remaining formaldehyde. Cells were then lysed and sonicated using a Diagenode bath sonicator at high setting with 30-s pulses over a 5-min period. Immunoprecipitation was carried out using the following antibodies: anti-acetyl-histone H3 (Millipore), ChIPAb+ Trimethyl-Histone H3(Lys4) (Millipore), ChIPAb+ Trimethyl-Histone H3(Lys27) (Millipore), anti-Histone H3 (di methyl K9) (Abcam), and rabbit IgG (Millipore). Real-time PCR analysis was carried out using the MJ Opticon 2 thermo cycler (MJ Research) and Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen). Cycling conditions were set up according to the manufacturer's instructions; annealing temperature for all primers was 55°C with 40 cycles of amplification. For ChIP primers and statistical analysis, see Supporting Information Method 2.