Cloning

For CRISPR experiments the sgRNA sequences are listed in (Table S1). These guide sequences were cloned into the pLentiCRISPRv2 vector (from Feng Zhang, Addgene plasmid #52961). All guide RNA sequences were confirmed by Sanger sequencing.

All cloning primers are listed in (Table S1). Human RhoA^G12V was amplified and N-terminal HA-tagged. This PCR product was used as a template for a second round of PCR amplification to add the Gateway adapter sequences. Human MRTFA was amplified out of the p3xFLAG-MRTFA vector (Addgene plasmid#11978) and tagged with gateway adapters which preserve the N-terminal 3x FLAG tag from the vector. The RhoA and MRTFA PCR products were first cloned into pDONR221 using the Gateway BP Clonase II Enzyme Mix from ThermoFisher (#11789020) using the manufacturer’s protocol. RhoA, MRTFA, and Gus (which is included in the BP reaction kit) were subcloned into the pLX301 lentiviral expression vector (from David Root, Addgene plasmid #25895) using the Gateway LR Clonase II Enzyme mix from ThermoFisher (#11791020). The presence of the correct insert in the final plasmid was confirmed by Sanger sequencing.