Simplechip universal qpcr mastermix

qPCR was performed using SimpleChIP Universal qPCR master mix (Cell Signaling Technology Inc.) and a primer pair (forward: 5’-TCAAGGCCAGCTTGGTCTAC-3’; reverse: 5’-CAGCAGCCCAGCAGCCTC-3’) flanking a region of the rat MHC class I gene RT1.A1 (Ensembl gene accession number: ENSRNOG00000038999) homologous to the silencer element of the “tissue-specific” region of the PD1 promoter used in the EMSAs. Each sample was run, in triplicate, in a QuantStudio 7 PRO Real-Time PCR System (Applied Biosystem, ThermoFisher Scientific, Waltham, MA, USA) and the immunoprecipitation efficiency was calculated using the percent input method.

PNA-treated or PNA-untreated K562 cells (4 × 10⁶ cells) were diluted to 0.5 × 10⁶ cells/ml and cross-linked with a final concentration of 1% formaldehyde (Sigma-Aldrich, 252549) for each IP. Chromatin was prepared and sheared according to manufacturer protocol using SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling, #9003) and QSONICA Q800R3 sonicator for nuclear lysis. For each condition, 10 μg of chromatin was incubated with 10 μg of anti-FLAG antibody (#14793, D6W5B, Cell Signaling) or 1 μg of normal rabbit polyclonal IgG control antibody (Cell Signaling, #2729) rotating overnight at 4°C. Chromatin was incubated with 30 μl of protein G magnetic beads (Cell Signaling, #70024) and washed, eluted, reverse cross-linked, and purified according to the manufacturer's protocol.
qPCR reactions were conducted in technical triplicate for each biological replicate for 2% input and IP samples using a StepOnePLUS Real-Time PCR System (Applied Biosystems), SimpleChIP Universal qPCR Mastermix (Cell Signaling, #88989), and HBB-IVS2 PNA target specific primers (Supplementary Table S1). Nontarget primer qPCR controls were also conducted for each replicate (Supplemental Fig. S7). Percent occupancy and fold enrichment values were calculated by percent input method from 2% input samples for each replicate.