Epitect chip oneday kit

Human PBMC were isolated by density gradient separation on Lymphoprep (Axis-Shield PoC As, Norway), and cultured in the presence of Concanavalin A (0.625 μg/ml, Sigma-Aldrich) and LPS (5 μg/ml, Sigma-Aldrich) for 72 h. Cells were then cross-linked and lysed according to EpiTect ChIP OneDay kit (Qiagen). After sonication to shear the chromatin, cellular debris was removed by pelleting. After pre-clearing the chromatin, 1% of the sample was removed as “input fraction”. The rest of the sample was incubated with either 2 μg anti-Bcl-6 [85 (link), 86 (link)] (N3, Santa Cruz Biotechnology), or anti-Survivin [87 (link)] (10811, Santa Cruz Biotechnology). In each experiment, one sample with unspecific antibody was included as negative control for nonspecific binding. A known Bcl-6-targeted sequence within the first non-coding exon of the Bcl-6 gene was amplified and used as an internal positive control. The immune complexes were washed, the cross-links reversed and the DNA purified according to the EpiTect ChIP OneDay kit (Qiagen). The purified DNA was used as template in real-time amplification using different oligonucleotide pairs for p53 [52 (link)], Bcl-6 [86 (link)] and Blimp-1 [50 (link)]. PCR products were resolved on 2% agarose gels and visualized by ethidium bromide staining and quantified by the ChemiDoc equipment and Quantity One software (Bio-Rad Laboratories).

For ChIP-seq analysis, CD4⁺ cells isolated from 12 women were stimulated with concanavalin A (ConA, 0.625 μg/mL, Sigma-Aldrich), and lipopolysaccharide (LPS) (5 μg/mL, Sigma-Aldrich) for 72 h and pooled in 4 independent samples for DNA purification. The cells were cross-linked and lysed with the EpiTect ChIP OneDay kit (Qiagen), as recommended by the manufacturer. After sonication to shear the chromatin, cellular debris was removed by pelleting. After pre-clearing, 1% of the sample was saved as an input fraction and used as background for nonspecific chromatin binding. Pre-cleared chromatin was incubated with 2 μg of anti-survivin (10811, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The immune complexes were washed, the cross-links were reversed, and the DNA was purified with the EpiTect ChIP OneDay kit (Qiagen) as recommended by the manufacturer. The quality of purified DNA was assessed with TapeStation (Agilent, Santa Clara, CA, USA). DNA libraries were prepared with ThruPLEX (Rubicon) and sequenced with a Hiseq2000 sequencing system (Illumina) according to the manufacturer’s protocols. Bcl-files were converted and demultiplexed to fastq with bcl2fastq (Illumina).

Chromatin immunoprecipitations were performed according to conditions suggested by the manufacturer’s EpiTect ChIP OneDay kit (Qiagen, GA-101) with ChIP-grade antibodies to mouse H3K27me3 (Qiagen, GAM-9205, 1:50). Briefly, formaldehyde was added to cells to cross-link proteins to DNA, and the cells were lysed in 1.5 ml lysis buffer (50 mM HEPES, pH 7.5, 140 mM NaCl; 1 mM EDTA; 1% Triton X-100; 0.1% sodium deoxycholate; 0.1% sodium dodecyl sulfate). Cell lysates were sonicated at the set of 2 s on/15 s off for three rounds using a Bioruptor ultrasonic cell disruptor (Diagenode, Denville, NJ) to shear genomic DNA to an average fragment size of 150–250 bp. Of the sample, 1% was removed for use as an input control. ChIP was performed following protocol provided by EpiTect ChIP OneDay kit (Qiagen) using antibodies toward H3K27Me3 (Qiagen). Anti-RNA polymerase II and control IgG were used as positive and negative controls, respectively. After washing and de-crosslinking, the precipitated DNA was purified using a QIAquick PCR purification kit (Qiagen).