Cleancap mcherry mrna

Plasmid DNA encoding AmCyan fluorescent protein (pAmCyan1-C1) was purchased from Clontech Laboratories Inc., Mountain View, CA, USA. The plasmid was propagated in Subcloning Efficiency™ DH5α E. coli competent cells (Invitrogen, Thermo Fisher Scientific, Darmstadt, Germany), then isolated and purified using Qiagen EndoFree Plasmid Mega Kit (Qiagen, Hildesheim, Germany) as per manufacturer’s protocol. CleanCap^® mCherry mRNA was purchased from Tri-Link BioTechnologies LLC, San Diego, CA, USA.

Cardiac fibroblasts from ROSA^Cas9-EGFP mice were transfected with guide RNAs using Lipofectamine MessengerMAX (Thermo Fisher) according to the manufacturer’s instructions. Briefly, 3 days postisolation, CD45−CD31−CD90 + cells were FACS sorted and replated at about 10,000 cells/cm². After 6 days, when reaching 80–90% confluency, cells were incubated for 5 min with the RNA (1:50)–lipid (1:33) complex in Opti-MEM for 10 min at room temperature. Media was changed after 48 hr and cells were collected for RNA isolation at 72 hr. Two guide RNAs, designed and synthesized in-house (JAX Genetic Engineering Technologies facility), were used for each of the target genes (Figure 5—source data 1). CleanCap mCherry mRNA (TriLink Biotechnologies), and guide RNAs for GFP and scrambled guides were used as controls. Guide RNA for the GFP gene were same as in Li et al., 2014 (link).