Instrument: Illumina HiScanSQ
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Fixed or unfixed chromatin out of primary human macrophage lysates was sheared and histone-DNA or TF-DNA complexes isolated with antibody. Multiplex DNA libraries of both H3K4me3 as well as PU.1 bound DNA were generated using Illumina’s ChIP-Seq Sample Preparation Kit (Illumina; # IP-102-1001) and the Multiplexing Sample Preparation Oligonucleotide Kit (Illumina; # PE-400-1001) using approximately 10 ng DNA following the manufacturer’s instructions. Briefly, purified DNA ends were repaired using PNK and Klenow enzyme, followed by treatment with Klenow exo minus polymerase to generate a protruding 3′ A base used for adaptor ligation. Next, size selection of libraries was performed as follows: DNA libraries were agarose gel purified, DNA fragments with approximately 220 bp size excised and eluted using QIAquick Gel Extraction kit (Qiagen). After subsequent adapter ligation to the repaired ends, an amplification step was performed for 5 cycles with PCR primers 1.1 and 2.1 (Illumina; # IP-102-1001). During a second 13 cycles amplification step multiplex PCR primers were added to the DNA libraries to construct multiplex sequencing libraries. For PU.1 DNA libraries multiplex PCR primers were added directly after adapter ligation to the amplification mix and 18 cycles of amplification were performed. After amplification steps DNA was purified.