Orange chip kit

Chromatin immunoprecipitation (ChIP) experiments were performed by using the Orange ChIP kit (Diagenode, Belgium), according to the manufacturer’s instructions. Briefly, cross-linking between DNA and protein was fixed in homogenized endometrial tissues by adding formaldehyde (37%; Sigma, USA). Next, by adding glycine, the cross-linking reaction induced by formaldehyde was quenched. Sonication was used to fragment chromatin to an average DNA fragment size of 200–600 bp using the Bioruptor Sonication System (Diagenode, UCD 200 Bioruptor). One percent of the sheared chromatin was saved, as control input DNA (without adding any antibody), while the rest was incubated with anti-MeCP2 antibody (Abcam, UK) for immunoprecipitation. Real time quantitative PCR (qPCR) was used to analyze level of DNA methylation modifications of H19-DMR region with specific primer sets (Table 1). The primers were designed to amplify two different regions of H19-DMR. Data is reported based on the fold enrichment of different immunoprecipitated DNA relative to 1/100 dilution of input chromatin. The % input was determined using the following formula: % input = 2^{(CtP inputP – CtP ChIPP)} B B × Fd × 100%.

ChIP experiments were carried out using the Orange ChIP kit (Diagenode, Belgium) as described
before (15 (link)). Cross-linked chromatin obtained from
1×10⁵harvested cells was immunoprecipitated with
anti-β-actin (Sigma cat # A1978, USA) and anti-RNA polymerase II (AbcamR cat # Ab5408, UK)
antibodies. The precipitated DNA was analyzed by
real-time PCR using specific primers for promoters
of marker genes as listed in Table 1. Data were expressed as fold enrichment of DNA associated with
immunoprecipitated enriched-DNA relative to a
1/100 dilution of input chromatin.

Histone modifications of H3K9ac, H3K9me2/3,
H3K4me2 and H3K27me3 were analyzed on the
regulatory region of CDH1, in prostaspheres and parental
cells, using chromatin immunoprecipitation quantitative
PCR (ChIP-qPCR) technique. In this regards, Orange
ChIP kit (Diagenode, Belgium) was used according to
the manufacturer’s instruction. Briefly, chromatin derived
from 1×10⁵ cells was used for each immunoprecipitation
reaction. PCR amplification was performed on the
DNA recovered from the ChIP samples as well as the
respective total chromatin input by using primers listed
in Table S2 (See Supplementary Online Information at
www.celljournal.org). Next, immunoprecipitated DNA
was quantified by real-time PCR, in a 7500 Real-Time
PCR system. The data were expressed as a percentage of
input DNA associated with the immunoprecipitated DNA
relative to a 1/100 dilution of input chromatin.