Additional data file 1:

Provided is a video clip showing the 15-day-old untreated EBs.

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Additional data file 2:

Provided is a video clip showing the 15-day-old puromycin treated EBs.

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Additional data file 3:

Summarized are the RT-PCR conditions and primers used for the RT-PCR experiments.

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Additional data file 4:

Provided are lists of probe sets for the subclusters A and B, as identified in the hierarchical clustering of probe sets upregulated in α-MHC⁺ cells (Figure 5).

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Additional data file 5:

Part a provides the genes belonging to the GOTERM_CC categories 'myofibril, striated muscle thin filament, actin cytoskeleton', 'cytoskeleton' and 'myosin', and GOTERM_BP category 'cytoskeleton organization and biogenesis' that are upregulated in the α-MHC⁺ cardiomyocytes (intersection of upregulation in α-MHC⁺ cardiomyocytes [twofold, t-test P value < 0.01] compared with control cells in the 15-day-old EBs and compared with undifferentiated α-MHC ES cells). Part b provides the genes belonging to the GOTERM_MF categories 'voltage-gated ion channel activity' that are upregulated in the α-MHC⁺ cardiomyocytes (intersection of upregulation in α-MHC⁺ cardiomyocytes [twofold, t-test P value < 0.01] compared with control cells in the 15-day-old EBs and compared with undifferentiated α-MHC ES cells).

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Additional data file 6:

Part a provides genes belonging to the KEGG pathway 'oxidative phosphorylation' that are upregulated in α-MHC⁺ cardiomyocytes (intersection of upregulation in α-MHC⁺ cardiomyocytes [twofold, t-test P value < 0.01] compared with control cells in the 15-day-old EBs and compared with undifferentiated α-MHC ES cells) and a schematic of the KEGG oxidative phosphorylation pathway. Part b provides the genes belonging to the GOTERM_CC_5 categories 'mitochondrion', 'mitochondrial membrane' and 'mitochondrial electron transport chain', and GOTERM_MF_5 categories 'hydrogen ion transporter activity', 'NADH dehydrogenase (quinone) activity' and 'sodium ion transporter activity' that are upregulated in α-MHC⁺ cardiomyocytes (intersection of upregulation in α-MHC⁺ cardiomyocytes [twofold, t-test P value < 0.01] compared with control cells in the 15-day-old EBs and compared with undifferentiated α-MHC ES cells). Part c provides genes belonging to the GOTERM_CC category "fatty acid metabolism" that are upregulated in the α-MHC⁺ cardiomyocytes (intersection of upregulation in α-MHC⁺ cardiomyocytes [twofold, t-test P value < 0.01] compared with control cells in the 15-day-old EBs and compared with undifferentiated α-MHC ES cells).

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Additional data file 7:

Part a provides genes belonging to the GOTERM_BP_5 category 'enzyme linked receptor protein signaling pathway' that are upregulated in the α-MHC⁺ cardiomyocytes (intersection of upregulation in α-MHC⁺ cardiomyocytes [twofold, t-test P value < 0.01] compared with control cells in the 15-day-old EBs and compared with undifferentiated α-MHC ES cells). Part b provides genes that belong to the GOTERM_MF_5 category 'protein kinase activity' that are upregulated in α-MHC⁺ cardiomyocytes (intersection of upregulation in α-MHC⁺ cardiomyocytes [twofold, t-test P value < 0.01] compared with control cells in the 15-day-old EBs and compared with undifferentiated α-MHC ES cells). Part c provides genes belonging to the GOTERM_BP_5 categories 'negative regulation of Wnt receptor signaling pathway' and 'negative regulation of signal transduction' that are upregulated in α-MHC⁺ cardiomyocytes (intersection of upregulation in α-MHC⁺ cardiomyocytes [twofold, t-test P value < 0.01] compared with control cells in the 15-day-old EBs and compared with undifferentiated α-MHC ES cells). Part d provides genes belonging to the Biocarta pathway 'p38 mitogen-activated protein kinase signaling" that are upregulated in α-MHC⁺ cardiomyocytes (intersection of upregulation in α-MHC⁺ cardiomyocytes [twofold, t-test P value < 0.01] compared with control cells in the 15-day-old EBs and compared with undifferentiated α-MHC ES cells). Part e provides genes belonging to the KEGG pathway 'Calcium Signalling' that are upregulated in α-MHC⁺ cardiomyocytes (intersection of upregulation in α-MHC⁺ cardiomyocytes [twofold, t-test P value < 0.01] compared with control cells in the 15-day-old EBs and compared with undifferentiated α-MHC ES cells). Part f provides genes belonging to the GOTERM_BP_5 'Regulation of cell size' that are upregulated in α-MHC⁺ cardiomyocytes (intersection of upregulation in α-MHC⁺ cardiomyocytes [twofold, t-test P value < 0.01] compared with control cells in the 15-day-old EBs and compared with undifferentiated α-MHC ES cells).

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Additional data file 8:

Provided are lists of probe sets for the subclusters A, B, and C, as identified in the hierarchical clustering of probe sets downregulated in α-MHC⁺ cells (Figure 6). Probe sets are listed with the corresponding gene symbol and gene title.

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Additional data file 9:

Part a provides genes belonging to the KEGG pathway 'cell cycle' as well as to the GO BP terms 'M-phase', 'mitotic cell cycle', and 'regulation of cell cycle' that are downregulated in α-MHC⁺ cardiomyocytes (intersection of downregulation in α-MHC⁺ cardiomyocytes [twofold, t-test P value < 0.01] compared with control cells in the 15-day-old control EBs and compared with undifferentiated α-MHC ES cells). It also provides a schematic of the KEGG cell cycle pathway, indicating the downregulated genes. Part b provides genes belonging to the BIOCARTA pathways 'G1/S checkpoint' and 'G2/M checkpoint' that are downregulated in α-MHC⁺ cardiomyocytes (intersection of downregulation in α-MHC⁺ cardiomyocytes [twofold, t-test P value < 0.01) compared with control cells in the 15-day-old control EBs and compared with undifferentiated α-MHC ES cells). Part c provides genes belonging to the GOTERM_BP_5 categories 'positive regulation of programmed cell death' that are downregulated in α-MHC⁺ cardiomyocytes (intersection of downregulation in α-MHC⁺ cardiomyocytes [twofold, t-test P value < 0.01] compared with control cells in the 15-day-old control EBs and compared with undifferentiated α-MHC ES cells).

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Additional data file 10:

Provided are probe sets both differentially regulated by puromycin treatment in 15-day-old β-actin EBs (twofold, Student's t-test P value < 0.01) and transcripts upregulated in α-MHC⁺ cells (intersection of upregulation in the α-MHC⁺ cardiomyocytes [twofold, Student's t-test P value < 0.01] compared with the control cells in the 15-day-old EBs and in the undifferentiated α-MHC ES cells).

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Additional data file 11:

Provides is a list of probe sets differentially regulated in the puromycin treated 15-day-old β-actin⁺ EBs.

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Additional data file 12:

Provided is the raw data for the α-MHC experiments.

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Additional data file 13:

Provided is the raw data for the α-MHC experiments.

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Additional data file 14:

Provided is the raw data for the β-actin control experiments.

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