We report ChIP-seq for H3K36me3 that were performed as supplementary experiments to the U-DNA-Seq study (GSE126822). H3K36me3 ChIP-seq was conducted in raltitrexed (RTX) treated or non-treated (NT) HCT116 cells expressing the UNG inhibitor (UGI). We found that the applied RTX treatment did not result in major differences in the genome-wide distribution of the H3K36me3 histone marker. For wider context of the study, see the related publication. Overall design: For the H3K36me3 ChIP-seq experiments, cross-linked chromatin was isolated from non-treated and RTX treated HCT116 cells expressing UGI, and fragmented to approximately 100-500 bp fragments by sonication. Anti-H3K36me3 antibody (Cat. No. 4909T, Cell Signaling Technologies) was used for the IP, and samples were sequenced by PE 150 bp in a Novaseq 6000 platform. Reads were filtered and aligned to the reference human genome (GRCh38.d1.vd1, (https://gdc.cancer.gov/about-data/data-harmonization-and-generation/gdc-reference-file)) using BWA. Genome scaled coverage tracks and fold change over control tracks were calculated using deepTools package (bamCoverage and bigwigCompare tools, respectively). Genomic background data from the ENCODE (ENCFF489VMD) was used as control. Broad peaks were also called using MACS2. For details see the Supplementary file 3 of the related publication.