Illumina NovaSeq 6000 paired end sequencing; Bulk RNA-Seq to study the effects of CENP-A overexpression, p53 status, and X- irradiation treatment in MCF-10-2A cells
Bulk RNA-Seq to study the effects of CENP-A overexpression, p53 status, and X- irradiation treatment in MCF-10-2A cells
We grew MCF-10-2A TetOn-CENPA-FLAG-HA cells expressing either empty vector (p53-WT) or dominant-negative p53 (p53-DN) with 0X Dox (no Dox), 1X Dox (10 ng/ml), or 10X Dox (100 ng/ml) 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 manufacturerâ€™s instructions and confirmed the quality and quantity of the RNA by TapeStation and NanoDrop. mRNA library preparation was performed by the Next Generation Sequencing (NGS) platform at Institut Curie using the Illumina TruSeq Stranded mRNA kit