Analysis of the Role of NuMA (TDIF) in DNA repair Through Next Generation Sequencing Techniques [OGAP-seq]

Purpose: To assess the role of NuMA (TDIF) in DNA repair, NuMA (TDIF) ChIP-seq, 4sU-seq in hydrogen peroxide treated and untreated WT and NuMA (TDIF) KD cells, and OGAP-seq in hydrogen peroxide treated and untreated WT and NuMA (TDIF) KD cells Methods: 4sU-seq was carried out in WT and NuMA (TDIF) KD (using a doxycyclin inducible system) untreated and peroxide treated duplicate RPE cells. The data was mapped using STAR, the bams coverted into bigwigs and differential expression between the conditions carried out using DE-seq2. NuMA (TDIF) ChIP-seq and OGAP-seq data was mapped using BWA. Both sets of mapped data were subsequently used to generate bigwigs showing fold-change relative to inputs using MACS2 and to generate metagene profiles using the CGAT suite of bioinformatics tools. Both sets of data were also run through FeatureCounts to count reads overlapping promoter regions. Results: Through our 4sU-seq data we identified a set of genes whose expression upon exposure to oxidative stress is dependent upon the presence of NuMA (TDIF). NuMA (TDIF) ChIP revealed increased occupancy of NuMA (TDIF) at promoters, which was increased for NuMA (TDIF)-regulated genes, paused genes and early response genes. OGAP-seq in the presence and absence of NuMA (TDIF) demonstrated that in the absence of NuMA (TDIF) there is increased damage across gene bodies and particularly promoters. Conclusions: Our study indicates that NuMA (TDIF) plays a role in the protection from and response to oxidative damage. This protective effect appears to be particularly strong at promoters regions, and may differ between distinct subsets of gene categories. Identification of NuMA (TDIF) regulated genes by comparing levels of newly transcribed RNA through 4sU-seq in duplicate WT and NuMA (TDIF) KD untreated and peroxide treated cells. Assessment of NuMA (TDIF) binding within the genome through NuMA (TDIF) ChIP-seq in duplicate MRC5 cell samples. Investigation into the effects of NuMA (TDIF) KD upon levels of oxidative damage across the genome through OGAP-seq in duplicate WT and NuMA (TDIF) KD samples derived from RPE1 cells.