Auto ideal chip seq kit for transcription factors

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.

Samples were fixed and prepared using the BLUEPRINT Consortium protocol. In brief, cells were fixed with 1% w/v formaldehyde for 10 min and quenched using 125 mM glycine before washing with PBS. Samples were sonicated using a Bioruptor (Diagenode), final SDS concentration of 0.1% w/v for 9 cycles of 30 s ‘on’ and 30 s ‘off’, and immunoprecipitated using an IP-Star Compact Automated System (Diagenode). For H3-specific antibodies the Auto-Histone ChIP-seq kit protein A (Diagenode) and for CTCF antibody the Auto iDeal ChIP-seq Kit for Transcription Factors (Diagenode) were used with Diagenode antibodies listed in Supplementary Table 6.
Immunoprecipitated and input DNA were reverse cross-linked (65 °C for 4 h), treated with RNase and Proteinase K (65 °C for 30 min). DNA was recovered with Concentrator 5 columns (Zymo) and prepared for sequencing using MicroPlex Library Preparation Kit v2 (Diagenode). Libraries analysed using High Sensitivity Bioanalyzer chips (5,067–4,626, Agilent), quantified using qPCR Library Quantification Kit (Kapa Biosystems), pooled and sequenced with a 50 bp single-end protocol on an Illumina Hiseq 2500.