Project description:Gene expression was compared between hTERT-RPE1 cells and hTERT-RPE1 cells stably overexpressing mouse MFRP with an N-terminal GFP fusion.
Project description:Transcriptional profiling of human hTERT-RPE1 cell spheroids comparing Control siRNA transfected hTERT-RPE1 cell spheroids with those transfected with YAP1 siRNA.
Project description:Gene expression was compared between hTERT-RPE1 cells and hTERT-RPE1 cells stably overexpressing mouse MFRP with an N-terminal GFP fusion. RNA was prepared from hTERT-RPE1 cells overexpressing GFP-MFRP and control cells. Both conditions were done in triplicate. Affymetrix GeneChip Human Genome U133Plus2.0 arrays were used to interrogate changes in gene expression. Image data were quantified with Affymetrix Expression Console Software and normalized with Robust Multichip Analysis.
Project description:Transcriptional profiling of human hTERT-RPE1 cell spheroids comparing Control siRNA transfected hTERT-RPE1 cell spheroids with those transfected with YAP1 siRNA. Two-condition experiment, Control siRNA vs.YAP1 siRNA hTERT-RPE1 cell spheroids. Biological replicates: 1 Control siRNA, 1YAP1 siRNA transfected, independently grown and harvested. Bothreplicates per array.
Project description:The impact of depleting SAF-A (HNRNPU) on the genome-wide replication timing program in human hTERT-RPE1 cells was assessed by a single-cell replication timing analysis.
Project description:RNA-seq upon MYCN activation in the hTERT-immortalized MYCN retinal pigmented epithelial cell line (RPE1–MYCN-ER). The parental hTERT-immortalized retinal pigment epithelium (hTERT-RPE1) was used as a control as this cell line does not carry the MYCN:ER expression construct. Both cell lines were treated with tamoxifen (400nM; 4-OHT) for MYCN induction (with 4-OHT or not). Analysis was performed 24 h, 48 h and 72 h upon MYCN activation, including four biological replicates per condition.
Project description:Mapping replication timing in the RPE1 and HCT116 cell lines revealed differences in replication dynamics and replication domain structures in the two cell lines
Project description:Chromosome segregation errors have been linked to DNA damage and genomic rearrangements. Accumulating evidence has shown that catastrophic genomic rearrangements, like chromothripsis, can result from lagging chromosomes undergoing aberrant DNA replication and DNA damage in micronuclei. Detailed characterization of genomic rearrangements resulting from DNA damage in micronuclei has been hampered because of difficulties in culturing daughter cells with DNA damage. Here, we employ a method by which a specific single chromosome is trapped in a micronucleus, followed by transfer to an acceptor cell. Next, stably propagating clonal cell lines with an extra chromosome were established and analyzed by copy number profiling and whole genome sequencing. While non-transformed, p53 proficient and telomerase-immortalized RPE1 cells showed a stable genome following addition of the transferred chromosome, we observed frequent de novo genomic rearrangements in cells derived from the HCT116 colorectal cancer cell line after chromosome transfer. The de novo rearrangements varied from simple deletions and duplications to complex rearrangements. Phase-informative SNPs revealed that the rearrangements specifically occurred on the transferred chromosome. We found that the complex rearrangements recapitulated all features of chromothripsis, including massive oscillation between two copy number states, localization to a single chromosome, random joining of chromosome fragments and non-homologous or micro-homologous repair. We describe an approach that enables the isolation of clonal cell lines with genomic rearrangements and chromothripsis on a specific chromosome in p53 proficient cells. The procedure enables further investigation of the exact mechanism leading to chromothripsis and the analysis of its consequences for cell survival (viability) and cancer development. We analyzed 38 cell clones, originating from HCT116 or RPE1 cells respectively, with Illumina beadchip arrays to test for unique de novo copy number variants and to determine the chromosome affacted by the CNAs.
