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Supplementary Materials a Comprehensive Analysis of Gene Expression Changes in a High Replicate and Open-Source Dataset of Diff Supplementary Materials A comprehensive analysis of gene expression changes in a high replicate and open-source dataset of differentiating hiPSC-derived cardiomyocytes Tanya Grancharova1,2, Kaytlyn A. Gerbin1,2, Alexander B. Rosenberg3,4, Charles M. Roco3,4,5, Joy Arakaki2, Colette DeLizzo2, Stephanie Q. Dinh2, Rory Donovan-Maiye2, Mathew Hirano3, Angelique Nelson2, Joyce Tang2, Julie A. Theriot2,6, Calysta Yan2, Vilas Menon7, Sean P. Palecek8, Georg Seelig3,9, Ruwanthi N. Gunawardane2* 1Authors contributed equally 2Allen Institute for Cell Science, Seattle, WA 3Department of Electrical & Computer Engineering, University of Washington, Seattle, WA 4Parse Biosciences, Seattle, WA 5Department of Bioengineering, University of Washington, Seattle, WA 6Department of Biology and Howard Hughes Medical Institute, University of Washington, Seattle, WA 7Department of Neurology, Columbia University Irving Medical Center, New York, NY 8 Department of Chemical and Biological Engineering, University of Wisconsin - Madison, Madison, WI 9Paul G. Allen School of Computer Science & Engineering, University of Washington, Seattle, WA * corresponding author, [email protected] Supplementary Figure S1 A Protocol 1 Protocol 2 D0 (undifferentiated) D12 D24 D90 D14 D26 WTC-11 TOMM20 TNNI1 WTC-11 TOMM20 TNNI1 WTC-11 TOMM20 TNNI1 WTC-11 TOMM20 TNNI1 WTC-11 TOMM20 TNNI1 WTC-11 TOMM20 TNNI1 Diff Exp1 1 1 1 1 Diff Exp2 1 1 1 1 1 1 Diff Exp3 1 1 1 1 1 1 1 1 Diff Exp4 2 1 2 1 2 1 2 1 Diff Exp5 1 2 1 1 1 2 1 2 1 3 1 2 Diff Exp6 2 1 Diff Exp7 2 Undiff 1 1 Protocol 1 Protocol 2 B D0 (undifferentiated) D12 D24 D90 D14 D26 WTC-11 TOMM20 TNNI1 WTC-11 TOMM20 TNNI1 WTC-11 TOMM20 TNNI1 WTC-11 TOMM20 TNNI1 WTC-11 TOMM20 TNNI1 WTC-11 TOMM20 TNNI1 Diff Exp1 1 1 1 1 Diff Exp2 1 1 1 1 1 1 Diff Exp3 1 1 1 1 1 1 1 1 Diff Exp4 2 1 2 1 2 1 2 1 Diff Exp5 1 2 1 1 1 2 1 2 1 3 1 2 Diff Exp6 2 1 Diff Exp7 2 Undiff 1 1 C Protocol 1 Protocol 2 D0 (undifferentiated) D12 D24 D90 D14 D26 WTC-11 TOMM20 TNNI1 WTC-11 TOMM20 TNNI1 WTC-11 TOMM20 TNNI1 WTC-11 TOMM20 TNNI1 WTC-11 TOMM20 TNNI1 WTC-11 TOMM20 TNNI1 Diff Exp1 1 1 1 1 Diff Exp2 1 1 1 1 1 1 Diff Exp3 1 1 1 1 1 1 1 1 Diff Exp4 2 1 2 1 2 1 2 1 Diff Exp5 1 2 1 1 1 2 1 2 1 3 1 2 Diff Exp6 2 1 Diff Exp7 2 Undiff 1 1 D D0 D12 D12 D24 D24 D90 E D12 D12 D14 D14 D24 D24 D26 Supplementary Figure S1. Sample overview A. Table summarizing all scRNA-seq samples. Each row represents one differentiation experiment (plates/samples set up on one day; see Fig. 5B). Numbers in the table indicate the number of wells that were collected. Wells were not pooled. B. Table highlighting scRNA-seq samples used in Figs. 1-3, Supplementary Figs. S2-S4, focusing on gene expression during cardiomyocyte differentiation with Protocol 1 at D0, D12, D24 and D90. C. Table highlighting scRNA-seq samples used in reproducibility analysis (Figs. 4-5, Supplementary Figs. S6-S7). D. UMAPs showing each of the samples from D0, D12, D24, D90 Protocol 1 analysis (D0 n = 2 samples; D12 n = 9 samples; D24 n = 9 samples; D90 n = 5 samples). Each individual sample highlighted in green in B is shown in a separate UMAP in red. The UMAP is the same as in Fig. 1. Box colored by time point (colors as in Fig. 1D) is drawn around all samples from a given time point. E. UMAPs showing each of the samples from D12, D14, D24, D26 reproducibility analysis (D12 n = 14 samples; D14 n = 14 samples; D24 n = 9 samples; D26 n = 11 samples). Each individual sample highlighted in green in C is shown in a separate UMAP in red. The UMAP is the same as in Fig. 4. Box colored by time point (colors as in Fig. 4C) is drawn around all samples from a given time point. Supplementary Figure S2 A y a Cluster Cluster D FN1 COL3A1 COL1A2 COL1A1 POSTN THBS2 OGN GPC5 EYS FSTL5 RP5−964N17.1 FLT1 KDR EGFL7 ESM1 ELTD1 MFAP4 IGF2 H19 A2M C7 GATA4 ZFPM2 KCNIP4 MYH6 ACTC1 TTN TNNT2 GPR126 AFP SERPINA1 LRP2 LINC00842 SBSPON KCNMA1 PRTG RP11−175E9.1 GABRP GRHL2 CNTN1 DCT TRPM3 ADAMTSL3 TYRP1 SERPINF1 ELN SULF1 BNC2 PCDH9 GRIA4 RELN RMST CPAMD8 CENPF MKI67 PDZRN4 CNTN5 CTNNA2 10 4 11 12 Day 3 13 D12 4 D24 2 5 D90 6 1 7 8 0 9 −1 B CTNNA2 C OGN 10 10 0 1 2 3 4 0 1 2 3 4 5 5 C11 0 0 UMAP2 UMAP2 C5 -5 -5 -10 -10 -10 -5 0 5 10 -10 -5 0 5 10 UMAP1 UMAP1 D TRPM3 E FN1 10 10 0 1 2 3 4 5 0 2 4 6 5 5 C11 0 0 UMAP2 UMAP2 C5 -5 -5 C7 -10 -10 -10 -5 0 5 10 -10 -5 0 5 10 UMAP1 UMAP1 Supplementary Figure S2: Non-cardiomyocyte cell types identified in D12, D24, and D90 samples generated with differentiation Protocol 1 A. Heatmap showing top differentially expressed genes from each pairwise cluster comparison between pairs of non-cardiomyocyte clusters (non-cardiomyocytes were identified by the absence of marker TNNT2; see Fig. 1C-D). Heatmap includes cardiomyocyte cluster 7, proliferative cardiomyocyte cluster 12, and all differentiated non-cardiomyocyte clusters. Normalized transcript abundance was centered and scaled across each gene (z-score color scale to the right of heatmap; red = standard deviations above mean; blue = standard deviations below mean; white = mean; for visualization purposes, 4 was set as the maximum z- score, and z-scores > 4 were set to 4). The dendrogram is based on hierarchical clustering of genes. Each row corresponds to one cell. B. UMAP from Fig. 1 colored by transcript abundance of CTNNA2, highlighting non-cardiomyocyte cluster C11 (orange shading in A and outline on UMAP). Increased red shading reflects higher levels of transcript. C. UMAP from Fig. 1 colored by transcript abundance of OGN, highlighting non-cardiomyocyte cluster C5 (mint green shading in A and outline on UMAP). D. UMAP from Fig. 1 colored by transcript abundance of TRPM3, highlighting non-cardiomyocyte cluster C11 (orange shading in A and outline on UMAP) and cardiomyocyte cluster C7 (purple shading in A and outline on UMAP). E. UMAP from Fig. 1 colored by transcript abundance of FN1, highlighting non-cardiomyocyte cluster C5 (mint green shading in A and outline on UMAP). Supplementary Figure S3 A B PRKG1 Day H19* Day LINC00881 EFNA5 PAM ACTG1 D24 PRSS35 D12 EGR1 PLN PDE3A D90 MYH7 D24 CRYAB SPHKAP PDE1C PDGFD C7 FHOD3 CPNE5 FBXL7 A2M MLIP IGF2 RNF150 H19* 4 INPP4B NLGN1 STK39 PLCL1 GOLIM4 4 HDAC9 3 SV2C RGS6 HS3ST4 FGF12 SOX6 CNTN5 2 2 MOXD1 CALD1 WWOX MFAP4 DOK4 SULT1E1 1 GRIN2A 0 NCKAP5 FAM19A4 HECW2 0 MYH6* STK38L BMPER* PRRX1 −2 LDLRAD4 COL2A1* −1 MYO1D NFIB PRTG* TRIM24 KCNQ5 MEF2C −4 −2 RNA28S5 ALPK2 GAPDH DENND5B ADAMTS12 WNT5B CCDC141 RALYL FBN2 MYH6* VCAN SC5D ATP1A1 RHOBTB3 ACTA1 PRTG* MASP1 BMPER* FRMD4B SNHG14 RBFOX2 COL2A1* TENM4 SMADs, TFs Signaling C D C2 C0 C1 C3 C2 C0 C1 C3 Max value wound healing Max value viral transcription PDE7A 3 LDLRAD4 3.3 viral gene expression translational initiation rRNA metabolic process PDE4D 4.2 RNA localization SMAD9 3 RNA catabolic process 3.4 ribonucleoprotein complex biogenesis ADCY5 regulation of transporter activity MEF2C 3.6 regulation of the force of heart contraction PDE3A 4 regulation of system process regulation of membrane potential TGFB2 4.1 regulation of ion transmembrane transporter activity PDE10A 3.7 muscle system process muscle hypertrophy in response to stress TGFBR2 2.7 3.7 multicellular organismal signaling PDE1C heart morphogenesis heart development extracellular matrix organization Ion channels Metabolic establishment of protein localization to endoplasmic reticulum C2 C0 C1 C3 C3 Max value C2 C0 C1 C3 Max value embryonic morphogenesis DNA conformation change CACNA1C 5.2 SLC2A3 3.3 cotranslational protein targeting to membrane cell-substrate adhesion CACNA1D 4.6 cardiac muscle hypertrophy in response to stress SLC27A6 3.7 cardiac muscle hypertrophy CASQ2 3.3 cardiac muscle adaptation cardiac conduction FABP3 3.4 6.1 actin-mediated cell contraction KCNIP4 KCNT2 3.3 D0v12 IGFBP5 4.4 D12v24 D24v90 SCN5A 3.2 100 150 200 250 50 Gene count KCNQ1 3.1 Supplementary Figure S3: Gene changes in cardiomyocyte populations over time A. Heatmap of the top 40 ranked genes from feature selection analysis between D12 and D24 cardiomyocytes. Genes that overlap between (A) and (B) are marked with an “*”. B. Heatmap of the top 40 ranked genes from feature selection analysis between D24 and D90 cardiomyocytes. Genes that overlap between (A) and (B) are marked with an “*”. C. Enriched gene ontology (GO) categories were identified for differentially expressed genes between Day 0 (C2) and Day 12 (C0), Day 12 (C0) and Day 24 (C1), and Day 24 (C1 and Day 90 (C3). Size of the circle represents the number of differentially expressed genes in each category. Top 10 enriched GO categories (ranked by multiple testing adjusted p-value) are shown for each pairwise comparison. See Supplemental Table S4. D. Functional gene categories that change between D24 and D90 cardiomyocytes. Transcript abundance distributions are shown for C2 (D0), C0 (D12), C1 (D24), and C3 (D90). Max value = maximum value of log1p normalized counts; dot = median.
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