Shearing chip kit

ChIP assay was performed using 1 Day ChIP kit and Shearing ChIP kit (Diagenode, Denville, USA) according to the manufacturer’s protocol. After lentiviral transduction and cross-linking, mouse kidney fibroblasts were fixed with formaldehyde and the DNA was sheared into small fragments. After incubation with a Myc-tag antibody (Cell signaling, Beverly, USA), the pulled-down complexes were de-crosslinked and treated with proteinase K. The purified DNA samples were proceeded further for library preparation by TruSeq RNA Library Prep Kit v2 (Illumina, USA) and quality control and library validation were performed by Fragment Analyzer and Kapa PCR (Illumina, USA), respectively. The fragments were sequenced by an Illumina HiSeq4000 instrument. For data analysis, a previous established protocol^{48 (link)} was followed. Briefly, the raw reads from two independent biological replicates were first concatenated and then peak calling was performed with MACSII in order to obtain the count numbers. We used settings for narrow peaks (200 bp window size, 200 bp gap size, and false discovery rate of 0.01) in all cases.

ChIP assay was performed using OneDay ChIP kit and Shearing ChIP kit (Diagenode, Denville, USA) according to the manufacturer’s protocol. After cross-linking, cells were fixed in formaldehyde and DNA was sheared to small fragment sizes. After incubation with specific antibodies against H3K9me3 (Abcam) and IgG as a negative control (Diagenode, Denville, USA), reversed cross-linking and proteinase K treatment were carried out. 5 μl of eluted DNA was added to the reaction mixture containing the primer pair (300 nmol/l each) (Supplemental Table 3), a ROX passive reference dye (Bio-Rad, Hercules, USA) and diluted 2× SYBR green Supermix (Bio-Rad, Hercules, USA) in a final volume of 25 μl for each PCR reaction. The real-time PCR reactions were performed in a 96-well reaction plate using the Mx3000P QPCR System (Stratagene, Santa Clara, USA). PCR reaction was stopped when the fluorescent signal increased over the threshold and electrophoresis of PCR products were done on a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, USA) according to the manufacturer’s protocol. Electrophoresis results are shown as virtual gel images as described in our previous publications72 (link)73 (link)74 (link).

Chromatin was isolated and immunoprecipitated using the Shearing ChIP Kit (Diagenode, C01020012, Denville, NJ, USA) and the OneDay ChIP kit (Diagenode, C01010080, Denville, NJ, USA), respectively, according to the manufacturer’s protocol. To immunoprecipitate H3K4me3 (Abcam, ab8580, Waltham, MA, USA), H3K9me3 (Abcam, ab8898, Waltham, MA, USA), H3K27me3 (Millipore, 07449, Darmstadt, Germany), H4K20me3 (Abcam, ab9053, Waltham, MA, USA), CTCF (Millipore, 07729, Darmstadt, Germany), and RNA Pol-II (Abcam, ab5408), we used 4 µg of each antibody and incubated them overnight. Immunoprecipitated DNA was analyzed by qPCR with the StepOne Real-time PCR system (Thermo Fisher Scientific, 4376600, Waltham, MA, USA) using specific primers for each locus. Total chromatin input from every sample was used as a reference for the comparative curves of the analyzed marks. Data were normalized against IgG.