ena-STUDY-INSTITUT CURIE-17-11-2020-15:59:01:183-535
Bulk RNA-Seq to study the effects of CENP-A overexpression, p53 status, and X- irradiation treatment in MCF10-2A cells
We performed bulk RNA-Seq to investigate global transcriptional changes upon overexpression of the centromeric H3 variant CenH3/CENP-A in p53 wild-type and defective cells, and after X-irradiation treatment. We established clonal MCF10-2A TetOn-CENPA-FLAG-HA cell lines where CENP-A can be reversibly induced by Doxycycline (Dox) treatment. Upon CENP-A overexpression, these cells exhibit different radiosensitivity depending on p53 status. In order to profile global changes in expression, we grew MCF10-2A TetOn-CENPA-FLAG-HA cells expressing either empty vector (p53-WT) or dominant-negative p53 (p53-DN) with 0X Dox (no Dox), 1X Dox (10ng/ml), or 10X Dox (100ng/m) for 24h. At time 0, we irradiated one set of cells by X-ray generator (4gy) while a control set remained un-irradiated (0gy). 6h later, we extracted RNA for RNA-seq. All conditions tested in duplicate. We extracted RNA directly from culture dishes using the Qiagen RNeasy Mini kit according to the manufacturer's instructions and confirmed the quality and quantity of the RNA by TapeStation and NanoDrop. mRNA library preparation and sequencing were performed by the Next Generation Sequencing (NGS) platform, Institut Curie, using the Illumina TruSeq high input RNA kit and NovaSeq 6000 sequencer.
We performed bulk RNA-Seq to investigate global transcriptional changes upon overexpression of the centromeric H3 variant CenH3/CENP-A in p53 wild-type and defective cells, and after X-irradiation treatment. We established clonal MCF10-2A TetOn-CENPA-FLAG-HA cell lines where CENP-A can be reversibly induced by Doxycycline (Dox) treatment. Upon CENP-A overexpression, these cells exhibit different radiosensitivity depending on p53 status. In order to profile global changes in expression, we grew MCF10-2A TetOn-CENPA-FLAG-HA cells expressing either empty vector (p53-WT) or dominant-negative p53 (p53-DN) with 0X Dox (no Dox), 1X Dox (10ng/ml), or 10X Dox (100ng/m) for 24h. At time 0, we irradiated one set of cells by X-ray generator (4gy) while a control set remained un-irradiated (0gy). 6h later, we extracted RNA for RNA-seq. All conditions tested in duplicate. We extracted RNA directly from culture dishes using the Qiagen RNeasy Mini kit according to the manufacturer's instructions and confirmed the quality and quantity of the RNA by TapeStation and NanoDrop. mRNA library preparation and sequencing were performed by the Next Generation Sequencing (NGS) platform, Institut Curie, using the Illumina TruSeq high input RNA kit and NovaSeq 6000 sequencer.