Epitect chip qpcr primer

shRNA‐Transduced ATDC5 cells (approximately 5 × 10 6) were cross‐linked with 1% formaldehyde (10 min at room temperature). ChIP assays were performed using the ChIP‐Express Enzymatic Kit (Active Motif, Inc.) according to the manufacturer's instructions. Briefly, chromatin was enzymatically sheared to an average size of approximately 300 bp and incubated with 2 μg of control IgG (Active Motif, Inc.) or antibody specific to SOX9 (Abcam, Cambridge, UK), and 25 μL of protein G magnetic beads overnight at 4°C. After reversal of cross‐linking and protein digestion with proteinase K, immunoprecipitated DNA was purified with the MiniElute PCR Purification Kit (QIAGEN), and PCR was performed using a SYBR Green ROX Master Mix (QIAGEN) according to the following parameters: enzyme activation, 95°C for 2 min, and then 40 cycles of 95°C for 15 s and 60°C for 1 min. EpiTect ChIP qPCR primers (QIAGEN) for the putative SOX9 binding site were used to amplify the target enhancer region within the Acan (GPM10391229‐05 kb) and Col2a1 (GPM1046416 + 03 kb) genes. Each probe represented a pool of primers that amplify many different amplicons around that region. For Col2a1, this is a region +3 kb from the transcription start site (TSS; Active Motif, Inc.) and for Acan this is a region –5 kb from the TSS. Both regions include previously described SOX9 binding sites.

ChIP assays were carried out using EZ ChIP kit (Millipore) according to the manufacturer's protocol with some modifications. 293T cells were treated with Tm or Tg prior to cross-linking. DNA fragments at around 200-1000 bp were achieved by sonication with Microson Ultrasonic Cell Disruptor (Misonix). For IP, the indicated antibodies (i.e. anti-XBP-1 or anti-PCAF antibodies) were added to the sheared chromatin individually and incubated at 4°C overnight. The DNA/protein/antibody complex was then pulled down by protein G agarose and the DNA in the complex was purified using QIAquick PCR purification kit (Qiagen). Quantitative-PCR was performed to determine the relative amount of DNA that was immunoprecipitated by anti-XBP-1 or anti-PCAF antibodies in the presence of Tm or Tg. The primer pairs used to amplify the promoter regions of BiP, CHOP and EDEM genes include: BiP (5′-GATGGGGCGGATGTTATCTA-3′ and 5′-CTCTCACACTCGCGAAACAC-3′), CHOP (5′-GACACTACGTCGACCCCCTA-3′ and 5′-GGTTCCAGCTCTGATTTTGG-3′), and EDEM (Epitect ChIP qPCR primers, Qiagen). Cells treated with DMSO served as a negative control. For overexpression, MCF7 cells were co-transfected with the indicated expression vectors two days prior to cross-linking, followed by ChIP-quantitative-PCR as described earlier.

The truChIP Chromatin Shearing Kit (Covaris) was used to prepare chromatin. Chromatin was sheared into 200- to 700-bp fragments using a Covaris S2 instrument (duty cycle, 2 %; intensity, 3; 200 cycles per burst; 4 min). The IgG and NF-kB p65 antibodies (2A12A7, Thermo Fisher) were used for immunoprecipitation by the Quick Chip Kit (Imgenex SYBR Green Master Mix, (Appliedbiosystems) was used for qPCR to quantify precipitated DNA). The primers are from Qiagen EpiTect ChIP qPCR primers.