HCT116 MCM10 RNF4 Chromosomal Microarray Analysis
Description
For CNV analyses, cells were submitted to the UMGC for DNA extraction and chromosomal microarray analysis (CMA). Microarrays were carried out in HCT116 WT, MCM10+/- Clone 8, RNF4-/- Clone 3, MCM10+/-:RNF4-/- Clone 8-4, and MCM10+/-:RNF4-/- 8-4 complemented with HA-RNF4 Clone F for one biological replicate. CMA was performed with the Infinium CytoSNP-850K v1.2 Beadchip (Illumina), according to the manufacturer’s instructions. The array contains approximately 850,000 single nucleotide polymorphisms (SNPs) markers spanning the entire genome with an average probe spacing of 1.8 kb. The data was analyzed using GenomeStudio Data Analysis Software version 2.0.5 based on the reference human genome (hg19/GRCh37). To generate a list of all CNV regions (CNVRs) between two cell lines, R Statistical Software (version 4.1.2; R Core Team 2021) was used with the handyCNV R package (version 1.1.7). For the different cell line comparisons, each identified CNVR was manually verified as a true CNV event by inspecting both the B allele frequency and log R ratio plots at the specified genomic locus in GenomeStudio (Figure 5c-d). True CNV events show changes in both the B allele frequency and log R ratio plots which measure the genotypes and signal intensity, respectively, for each SNP marker in the Beadchip array.