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Illumina Infinium MethylationEPIC BeadChip (850k) data of NCCIT / 2102EP cells deficient for CD24 and parental controls

ABSTRACT: Illumina Infinium MethylationEPIC BeadChip (850k) data of NCCIT / 2102EP cells deficient for CD24 and parental controls

PROVIDER: PRJNA736308 | ENA |

REPOSITORIES: ENA

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Illumina Infinium MethylationEPIC BeadChip (850k) data of NCCIT / 2102EP cells deficient for CD24 and parental controls

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Project description:Illumina Infinium MethylationEPIC BeadChip (850k) array analysis of DNA methylation of primary CAF isolated from fresh ex vivo GCT patient tissue. Purity of CAF populations were verified by qPCR analysis of common CAF, GCT, endothelial and epithelial markers. 14 different CAF cultures from distinct GCT subtypes (seminoma(SE), non-seminoma(NS)) and 5 non-malignant fibroblast cultures were analyzed.

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Project description:Illumina Infinium DNA Methylation (5mC) profiling arrays are a popular technology to measure genome-scale distribution of 5mC at low cost and high throughput, especially in cancer and other complex diseases. Following the success of the HumanMethylation450 array (450k), Illumina released the MethylationEPIC array (850k) featuring increased coverage of enhancers in addition to regulatory regions primarily covered by the 450k (i.e. promoters, gene bodies). Despite its widespread use, the analysis of 850k data remains suboptimal as it mostly still relies on Illumina’s default annotation, which underestimates enhancers and long noncoding RNAs (lncRNAs). We thus developed an approach, based on ENCODE and LNCipedia databases, that greatly improves Illumina’s default annotation of enhancers and long noncoding transcripts. Comparisons between the re-annotated 850k and its precursor, the 450k, or RRBS, another high-throughput 5mC profiling technology, revealed that the 850k covers at least three times more enhancers and lncRNAs than the other two technologies. We further investigated the reproducibility of the three technologies and applied various normalisation methods to 850k data, showing that most of them reduce variability to a level below that of RRBS. When analyzed with our new annotation and normalization pipeline, profiling for 5mC changes in breast cancer biopsies with the 850k highlighted aberrant enhancers methylation as the predominant feature, confirming previous reports. In conclusion, our study provides an updated analysis pipeline for 850k data based on a refined probe annotation and normalization that allows for the improved analysis of methylation at enhancers and long noncoding transcripts.

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Project description:Type II testicular germ cell tumors (TGCT) are the most frequently diagnosed solid malignancy in young men. Up to 15 % of patients with metastatic non-seminomas show cisplatin resistance having a very poor survival rate due to lacking treatment options. Transcriptional cyclin-dependent kinases (CDK) have been shown to be effective targets in treatment of different types of cancer. Here, we investigated the effects of the CDK inhibitors dinaciclib, flavopiridol, YKL-5-124, THZ1, NVP2, SY0351 and THZ531. XTT viability assay revealed strong cytotoxic impact of CDK7/ 12/ 13 inhibitor SY0351 and CDK9 inhibitor NVP2 on the TGCT wild type cell lines (2102EP, NCCIT, TCam2) and the cisplatin resistant cell lines (2102EP-R, NCCIT-R). The CDK7 inhibitor YKL-5-124 showed strong impact on 2102EP, 2102EP-R, NCCIT and NCCIT-R cell lines leaving the MPAF control cell line mostly unaffected. FACS based analysis revealed mild effects on the cell cycle of 2102EP and TCam2 cells after SY0351, YKL-5-124 or NVP2 treatment. Molecular analysis showed a cell line specific response for SY0351 and NVP2 inhibition while YKL-5-124 induced similar molecular changes in 2102EP, TCam2 and MPAF cells. Thus, after TGCT subtype determination CDK inhibitors might be a potential alternative for optimized and individualized therapy independent of chemotherapy sensitivity.

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