mRNA sequencing of MDA-MB-231 engineered for doxycycline-inducible overexpression of wild-type or mutant myocardin related transcription factor A
Myocardin Related Transcription Factor A (MRTF-A), a co-factor or serum response factor (SRF), has been implicated in breast cancer cell migration and metastatic potential. The goal of this sequencing effort was to determine which mRNA were regulated by MRTF-A overexpression and which mRNA were differentially regulated when function-inhibited (SAP-domain deleted or SRF-binding deficient) mutant MRTF-A were overexpressed. GFP and luciferase expressing MDA-MB-231 cells engineered with doxycycline inducible overexpression of wild-type or mutant MRTF-A were treated with doxycycline for 72 hours prior to RNA collection. Two mutant versions of MRTF-A were used 1) a SAP-domain deleted mutant (deleted base-pairs 343-378) and 2) an SRF binding deficient mutant (mutated K237A, Y238A, H239A, Y241A). The cells were plated in a tissue-treated culture dish or on top of matrigel during doxycycline induction. RNA was extracted using the Qiagen RNeasy Plus Mini Kit according the manufacturer's instructions. Libraries were obtained starting from total RNA following the Illumina Stranded Total RNA preparation Ligation with Ribo-Zero Plus protocol. mRNA was then sequenced on the Illumina NextSeq2000.