Express one step sybr greener supermix kits

Chromatin Immunoprecipitation (ChIP) assays were performed as described earlier32 (link)33 (link). Cross-linking of HepG2 cells was done for 10 min in the cell culture medium with 1% formaldehyde at room temperature. 10⁶ HepG2 cells were used for each ChIP. The HepG2 cells were dissolved in 200 μl of sodium dedecyl sulphate (SDS) lysis buffer (10 mM EDTA, 50 mM Tris, pH 8.1, 1% SDS), and the sonication was performed to generate DNA fragments of 200–1000 bp. 10-fold dilution of chromatin was performed with ChIP dilution buffer (1.2 mM EDTA, 1.1% Triton X-100, 16.7 mM Tris, pH 8.1, 167 mM NaCl, 0.01% SDS). Chromatin was precipitated with their respective antibodies. Immunoprecipitated complexes of protein and DNA were reverse cross-linked and extracted with phenol/chloroform and precipitated with ethanol to get the purified DNAs.
The purified DNAs were amplified by QPCR using QuantStudio 6 Flex Real-Time PCR System (Life Technologies) and EXPRESS One-Step SYBR GreenER SuperMix Kits (Life Technologies). Primers for QPCR were reported2 (link) or designed and listed in Table 1.

The total RNA from the cells was extracted by TRIzol (Life Technologies) according to standard methods. 100 ng of the total RNAs were reverse-transcribed with random primers and 1^st-strand cDNA was prepared using First Strand cDNA synthesis Kit (Fermentas), and the synthesized cDNA was used as template for QPCR with EXPRESS One-Step SYBR GreenER SuperMix Kits (Life Technologies). The sequences of the used primers are listed in Table 1.