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We report a new immunoprecipitation-coupled sequencing (DIP-Seq) application termed U-DNA-Seq, where a tailored and catalytically inactive uracil-DNA glycosylase (UNG) was used as uracil-DNA sensor to immunoprecipitate uracil containing genomic DNA fragments. More...

We report a new immunoprecipitation-coupled sequencing (DIP-Seq) application termed U-DNA-Seq, where a tailored and catalytically inactive uracil-DNA glycosylase (UNG) was used as uracil-DNA sensor to immunoprecipitate uracil containing genomic DNA fragments. Genomic uracil was profiled in drug-treated (5-fluoro-2'-deoxyuridine (5FdUR) or raltitrexed (RTX)) or non-treated (NT) HCT116 cells expressing the UNG inhibitor (UGI). The same experiments were also performed in the mismatch repair proficient version of the HCT116 cells (HCT116MMR), where chromosome 3 is reinserted to restore functional MMR (PMID: 8044777). Moreover, wild-type HCT116 or K562 cells were also measured. We found that regions of uracil enrichment in this assay were rather broad as compared to the sharp peaks typical in ChIP-seq. Therefore, we applied an approach alternative to the conventional peak calling. Namely, we calculated genome scaled coverage tracks and log2 ratio tracks of the enriched versus the input samples using deepTools package (bamCoverage and bigwigCompare tools, respectively) to provide a more appropriate description of uracil-enriched genomic regions. Interval (bed) files were also derived from these log2 ratio tracks to be able to screen large datasets for colocalizing features with them. For wider context of the study, see the related publication. Overall design: Genomic uracil patterns were determined in drug-treated (5FdUR or RTX) HCT116 cells that are either deficient (HCT116) or proficient in mismatch repair (HCT116MMR) and stably express the UNG inhibitor protein, UGI (samples are as follows: 5FdUR_UGI_HCT116, RTX_UGI_HCT116, 5FdUR_UGI_HCT116MMR, RTX_UGI_HCT116MMR). These were compared to the non-treated UGI-expressing (NT_UGI_HCT116 and NT_UGI_HCT116MMR), as well as to the wild-type HCT116 (WT_HCT116) and K562 (WT_K562) cells. In case of both HCT116 and HCT116MMR samples two independent biological replicates were measured (rep1 and rep2), while in case of K562, only one experiment was done. Genomic DNA was isolated from each sample and fragmented to approximately 300 bp fragments by sonication (“input” samples (son)). A part of the input sample was incubated with FLAG-tagged uracil-DNA sensor, which was finally precipitated by anti-FLAG beads, resulting in the “enriched U-DNA” sample (IP). Control U-DNA-IP experiments (ctr) were also done using sensor-free beads in 5FdUR treated and non-treated HCT116. Input, U-DNA enriched, and control samples were sequenced by Illumina paired-end reads (PE) 100 bp in a HiSeq 4000 or 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-files)). Here we provide the following processed files for each pair of samples: 1) log2 ratio (enriched versus input) tracks for individual replicates as well as for merged data (log2.bw); 2) interval files of uracil enriched regions derived from the log2 ratio tracks (region.bed). Regarding the control experiments, log2 ratio (enriched corrected with scaled control versus input) tracks are provided as processed data. For details see the Supplementary Material of the related publication. Less...