Ideal chip seq kit for histones

ChIP-qPCR was performed using the iDEAL ChIP-seq kit for histones (Diagenode) according to the manufacturer's instructions. Briefly, cells (transfected with either CAG-19PS or scrambled ON2) were fixed in 1% formaldehyde for 8 min, and following chromatin extraction aliquots of one million cells, were sonicated for 12 cycles of 30 s on/off in 1.5 ml Bioruptor Microtubes (Diagenode) 1.5 ml tubes with a Bioruptor pico (Diagenode). One million cells were used per immunoprecipitation with 3 μg of antibodies against active histone modifications: histone 3 lysine 4 trimethylation (H3K4me3) (Ab-003-050, Diagenode) and histone 3 lysine 27 acetylation (H3K27ac) (ab4729, Abcam) or 5 μg of RNA Pol II ser2 antibody (ab5095, Abcam). DNA was isolated following reverse cross-linking with QIAquick PCR Purification Kit (QIAGEN). ChIP DNA was diluted for qPCR fifty times. Briefly 3 μl of diluted ChIP DNA was used per reaction, in triplicate with FastStart Universal SYBR Green Master (Roche). Primers are listed in Table 2.

ChIP was performed with the iDeal ChIP-seq Kit for Histones (Diagenode) following the provider’s instructions. Briefly, isolated DCs were fixed with formaldehyde (1%) for 10 min at 37°C, and reaction was quenched with cold glycine. Cells were lysed with radioimmunoprecipitation assay buffer 20 min at 4°C, and nuclei were pelleted at 2300g for 5 min. Later, nuclear lysis buffer was applied (130 μl), and chromatin was sheared by using a Covaris sonicator and the program “Duty 10% PIP” 175 cycles 200 for 15 min. Sheared chromatin was incubated with the corresponding anti-histone antibody or immunoglobulin G control (1.5 μg/ml) conjugated with magnetic beads overnight at 4°C. After incubation, beads were washed and DNA fragments were eluted and decross-linked for 8 hours at 65°C. Last, after fixation reaction was reversed (8 hours, 65°C), DNA was isolated with the iPure beads v2 as indicated by the manufacturer protocol. qPCR was performed using primers against already described promoter regions for the indicated genes (ChIP-seq) as well as against the open regions detected for those genes in the ATAC-seq (table S2). Control primers for glyceraldehyde-3-phosphate dehydrogenase promoter region were used as a positive control.

We used H3K27me3 and H3K27ac ChIP-seq data, and RNA-seq data, to evaluate the performance of HMCan-diff in two experiments: (i) lung adenocarcinoma (A549 cell line) compared to normal lung tissue; (ii) human breast adenocarcinoma (MCF7 cell line) compared to primary human mammary epithelial cells (HMEC cell line). The A549 data were generated using the Diagenode polyclonal antibody specific to H3K27me3 (C15410069) and the Abcam antibody specific to H3K27ac (ab4729). Data is deposited in GEO with accession number GSE75903. Chromatin preparation and ChIP were performed with the Ideal ChIP-seq kit for histones according to the supplier's protocol (Diagenode). Data for the lung tissue were obtained from the epigenomic roadmap database, and data for the MCF7 and HMEC cell lines were obtained from ENCODE.