Ip star compact

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.

The WGA products from Ampli-1, REPLI-g, DOPlify and the bulk sample were fragmented to an average size of 350 bp using the S2 Focused Ultrasonicator (Covaris, Woburn, USA) following the manufacturer’s instructions (Duty cycle of 10%, Intensity of 5 and 200 cycles/burst, Duration 45 sec). Between 350ng and 1 µg of WGA product was used as input for fragmentation. Sequencing libraries of the fragmented samples were prepared with the TruSeq DNA PCR-free HT library preparation kit (Illumina) on the IP-Star Compact (Diagenode, Seraing, Belgium). Library quantification was performed using a Sequencing Library qPCR Quantification kit (Illumina, San Diego, USA) to quantify the sequenceable DNA fragments containing the correct adapters (this was performed on the samples of all four methods). The libraries from the different samples were pooled equimolarly, denatured and diluted to a final loading concentration of 2.5pM for sequencing. Finally, single-end indexed 75 bp sequencing was performed on a high-output NextSeq500 flow-cell (Illumina, California, USA).