Evos m5000 epifluorescence microscope

Immunofluorescent staining of troponin (Abcam ab10214), and video documentation of beating cardiomyocytes validated successful CM differentiation (Video S1). Transfections of 21 days differentiated CMs were performed with 2x10⁶ cells and 10 μg of the MPRA plasmid pool (split in two wells of a 6-well plate) and Lipofectamine Stem Transfection Reagent (STEM00015, ThermoFisher) for 48 h. Transfections with GFP served to evaluate transfection efficiencies on an EVOS M5000 epifluorescence microscope (ThermoFisher, Figure S1).
Successful VSMC differentiation of hTERT-immortalized adipose derived primary human mesenchymal stem cells (MSCs, SCRC4000, ATCC) was validated by quantification of smooth muscle markers (transgelin [TAGLN], calponin-1 [CNN1], and smooth muscle actin [ACTA2]) using qRT-PCR with PowerUp (ABI) (Figure S1, Table S24).¹²⁴ (link) CTs were normalized to GAPDH as a housekeeper. 19-20 days differentiated VSMCs (1.1x10⁶ cells / 10 cm dish) were transfected with 12 μg MPRA oligo pool and 12 μl GeneXPlus (1:1 ratio, ACS-4004, ATCC) for 48 h. Transfection efficiency was evaluated as described for CMs (Figure S1). Lower transfection efficiencies and thus lower barcode recovery rates were observed in VSMCs, consequently impacting our statistical power to detect regulatory variants in this cell type.

Confocal imaging was performed on an Opera Phenix imaging system (PerkinElmer). For NRR analysis, pre-expansion images were acquired with a 63x water objective (NA 1.15) in stacks of 6 planes spaced 0.5 microns apart and post-expansion images of the same plate were acquired using a 20x water objective (NA 1.0) in stacks of 50 planes spaced one micron apart. Prior to expansion, 61 tiled fields at the center of each well were imaged at 63x to capture cells that would be incorporated into the gel. After expansion, 12 fields in a cross pattern were imaged at 20x to capture a representative set of images for each gel across multiple wells. As a caveat, the centers of gels in HiExM should be avoided in imaging as they often include cells that are torn and disfigured due to the process of expansion detaching the gels from the device. Movies S1 and S2 were obtained on an EVOS M5000 epifluorescence microscope (ThermoFisher Scientific). For cardiomyocyte experiments, images were acquired from 4 independent experiments. The sample sizes for each condition were as follows: 118, 111, 110, 113, and 77 cells for DMSO control, 1 nM, 10 nM, 100 nM, and 1 μM Dox, respectively across the four independent experiments.