ChIP-Rx Protocol for Genome-wide Histone Profiling

ChIP-Rx was performed with 1×10⁷ human cells and 1×10⁶ MEF cells for spike-in normalization as described in a previous study [21 (link)]. For immunoprecipitation, sonicated chromatin was incubated with 10 μg of specific antibodies and 15 μL of pre-blocked Protein A/G beads (Smart-Lifesciences). After extensive washes, the captured DNA was eluted for library preparation using the NEBNext Ultra II DNA library prep kit for Illumina before sequencing on a NovaSeq 6000. ChIP-Rx reads were aligned to the human genome (UCSC hg38) and mouse genome (mm10) with Bowtie2 version 2.2.6 [79 (link)], using parameters: --local --very-sensitive. The resulting reads were normalized with the aligned mouse reads. The aligned human BAM files were normalized and converted to bigwig files for visualization in the UCSC Genome Browser. Peaks were called using MACS2 version 2.1.2 with default parameters [81 (link)]. MEME-ChIP [82 (link)] was used to further analyze peak region motifs. Heatmap and metaplots were made for the indicated windows using the average coverage (r.p.m.).