Superarray chip qpcr data analysis template

ChIP assays were performed using an EZ-ChIP kit (Millipore) according to the manufacturer’s instructions. Briefly, cells were crosslinked with 1% formaldehyde for 10 min at room temperature, and the formaldehyde was then inactivated by addition of 125 mM glycine. Chromatin extracts containing DNA fragments were immunoprecipitated using specific antibodies. The ChIP-enriched DNA was then uncrosslinked and subjected to quantitative PCR. Data were analyzed using the SuperArray ChIP-qPCR Data Analysis Template (Qiagen/SABiosciences) [55 (link), 56 (link)]. The primers and antibodies used in these experiments are shown in Supplementary Tables 1 and 2.

Immunoprecipitated DNA was used as a template for real time ChIP qPCR analysis using EpiTect ChIP Custom qPCR Arrays (Qiagen). Genes were chosen based on RNA-seq day one post-challenge IPA analysis. Primer sequences for regions 1kb upstream of the transcription start site of IRF-1, iNOS, GBP2, GBP5, GBP6, CXCL9, CXCL10, CXCL11, SOCS-1, CIITA, IFIT2, and IFI47 were used in the array. A master mix consisting of 2ul DNA per 25ul reaction was mixed with RT² SYBR Green qPCR master mix (Qiagen) according to manufacturer’s recommendations and Real Time ChIP qPCR arrays were performed using 7300 real-time PCR System (Applied Biosystems, Foster City, CA). Percent IP was calculated based on comparative threshold values using the SuperArray ChIP-qPCR Data Analysis Template supplied by Qiagen according to manufacturer’s instructions.