Resting and proliferating Tregs were sorted based on cell trace violet dilution. Total RNA was isolated from these cells by using RNAeasy columns (Qiagen). The cDNA synthesized from total RNA was used for RT-qPCR analysis with Fast SYBR green master mix (Applied Biosystems) and gene specific primers (listed in supplementary table-1 ) by using AB ViiA7 RT-qPCR instrument (Applied Biosystems). Gene expression values were calculated by comparative ΔCt method after normalization to GAPDH internal control and expressed as fold change over respective controls.
Micro array analysis was performed in duplicate using the Affymetrix GeneChip Mouse Genome 430 2.0 microarray at Center for genomics core facility, University of Illinois at Chicago. Briefly, biotinylated cDNA was synthesized from total RNA using biotinylated dNTPs and allowed to hybridize with microarrays and scanned. Arrays which passed quality control tests were further subjected to gene expression analysis after normalization with housekeeping gene controls. Data were analyzed using the R-package software. Student’s t-test was used to filter differentially expressed genes Micro array has been submitted to NCBI-Gene Expression Omnibus database and publicly available (Accession No. GSE81051).
Micro array analysis was performed in duplicate using the Affymetrix GeneChip Mouse Genome 430 2.0 microarray at Center for genomics core facility, University of Illinois at Chicago. Briefly, biotinylated cDNA was synthesized from total RNA using biotinylated dNTPs and allowed to hybridize with microarrays and scanned. Arrays which passed quality control tests were further subjected to gene expression analysis after normalization with housekeeping gene controls. Data were analyzed using the R-package software. Student’s t-test was used to filter differentially expressed genes Micro array has been submitted to NCBI-Gene Expression Omnibus database and publicly available (Accession No. GSE81051).