Auto ideal chip seq kit for histones

HeLa cells were exposed to 44 °C for 1 h and immediately fixed for 10 min with 1% formaldehyde (Carl Roth Inc) at RT followed by 5 min blocking with 125 mM Glycine at RT. Chromatin was extracted using the truChlP Chromatin Shearing Kit (Covaris, #520154) according to the manufacturer´s instructions. The chromatin was sheared by sonication to a DNA fragment size of 200–600 bp and precipitated using an antibody against human BRD4 (Bethyl Laboratories, A301-985A100, RRID:AB_2620184). ChIPs were run on the IP-Star compact system using the Auto iDeal ChIP-seq kit for histones (Diagenode, #C01010051) according to the manufacturer’s recommendations for ChIP preparation. ChIP-DNA was sequenced on a HiSeq4000, 50-bp single-end. Reads were mapped to the hg19 genome using bwa-0.7.12 with default parameters^{28 (link)}. Peaks were called with MACS2^{29 (link)} with the parameters “bw 500 -mfold 2100 -broad broad-cutoff 0.1 –bdg”. For visualization the bigwig files were converted to bedgraph files that were used for visualization with R/Bioconductor GViz package ^{30 (link)}.

Before chromatin immunoprecipitation (ChIP), isolated monocytes were cross-linked for 10 minutes at 18°C using formaldehyde at a final concentration of 1%. Fixation of DNA-protein links was stopped with 0.125 M glycine. After cell lysis (10⁶ cells per 200 μl lysis buffer), DNA-shearing was conducted in the Bioruptor Pico^® (Diagenode, Liège, Belgium) to achieve fragment sizes of 150–200 base pairs. Immunoprecipitation, reverse cross-linking and DNA purification were performed using an automated system (IP-Star^® Compact) with specific reagent kits (Auto iDeal ChIP-seq Kit for Transcription Factors, Auto iDeal ChIP-seq Kit for Histones) as well as antibodies against CTCF, trimethylated lysine 27 of histone 3 (H3K4me3) and acetylated lysine 27 of histone 3 (H3K27ac, all obtained from Diagenode, Liège, Belgium) according to manufacturer`s instructions. The amount of obtained ChIP-DNA (diluted in pure H₂O) was quantified using a Qubit^™ assay (Qubit Fluorometric Quantitation, Thermo Fisher Scientific, Waltham, USA). Subsequent quantitative PCR analysis was done using a StepOnePlusTM PCR-cycler (Applied Biosystems, Foster City, USA) as well as specific primer sets for CM 1–10 as previously identified by Majumder and Boss [22 (link)] or primer sets for the promoter regions of classical HLA class II genes. ChIP-antibodies and primer sequences are listed in Tables B and C inS4 Table.

Cells were cross-linked in formaldehyde at a final concentration of 1% for 8 min. All experiments reagents were included in the Auto iDeal ChIP-seq kit for Histones (Diagenode, Liege, Belgium). Sonication was performed using a Bioruptor Plus sonication devise (Diagenode, Liege, Belgium) under the optimal conditions to shear cross-linked DNA to fragments if 100–300 base pairs in length. ChIP was conducted with the IPStar Compact Automated System (Diagenode, Liege, Belgium). ChIP were performed starting from 1 million cells per IP, using the indirect method in 200 μL final volume. Crossed-linked DNA was incubated 13 h with the antibody (H3K27me3 catalog number C1540195 or H3K4me3 catalog number C15410003) and 3 h with the beads. After 5 min washes, eluates were recovered and reverse cross-linked for 4 h at 65 °C. Samples were treated for 1 h with RNAse at 37 °C, prior to DNA purification with the Auto IPure kit v2 (Diagenode, C03010010). Libraries were performed using NEBNext Ultra Library Prep Kit for Illumina (New England Biolabs). Sequencing was performed with Illumina NextSeq500 technology (Helixio, Clermont-Ferrand, France) using the following parameters: single-read, 50 bp, 40 million reads.