Chromatin Immunoprecipitation (ChIP) qPCR Assays

Chromatin Immunoprecipitation assays were performed using MAGnify TM Chromatin Immunoprecipitation System (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Specific anti-acetyl histone H3 (Abcam) and anti-acetyl histone H4 (Abcam) were used to determine the promoter acetylation of perforin and granzyme-B. Normal rabbit IgG was used as negative control. DNA was extracted and analyzed by quantitative real time PCR (qPCR) with specific primers for perforin (region I forward:5′-GATGAGGGCTGAGGACAG-3′; region I reverse:5′-TCTTCACCGAGGCTCCTG-3′; region II forward:5′-CTGCTGGCCTGTTCATCAAC-3′; region II reverse: 5′-CTGTCCTCAGCCCTCATC-3′) and granzyme B (region I forward: 5′-GGGTGGGCAGCATTTACAG-3′; region I reverse: 5′-TTCTCAGGAAGGCTGCCC-3′; region II forward: 5′-CACTTCATAGGCTTGGGTTCC-3′; region II reverse: 5′-CCTCTGGTTTTGTGGTGTCTC-3′). 1% of starting chromatin was used as input. Relative data quantification was performed using 2^−ΔΔCt method, using formula: % Input = 2 (Ct Input − Ct ChIP) × Input dilution factor × 100 and expressed in the form of % input as described earlier (38).