## 2 Results  
### scRNAseq of Retinas Prepared from Control and T3-Treated Mice  
To obtain transcriptomes of individual cells in the retina after the stimulation of TH signaling, droplet sequencing was conducted in the retinas of control mice and mice that were treated with T3 (20 ug/mL via drinking water) for 4 weeks. This treatment increases the serum T3 level by about 5-8-fold [37]. Retinas from four mice in each group were used. Retinal cells were dissociated/isolated using cell dissociation medium, and the average cell viability was about 57.5% according to flow cytometry. Following preprocessing and quality control, we identified 155,866 single cells that were grouped into 13 clusters with 918 median genes and 1465 median unique molecular identifiers per cell. The clusters included cells from both the control and T3-treated retinas. The clusters were annotated using a curated set of known marker genes (Table 1) representing different types of retinal cells. The cluster analysis showed that rods represent 76.0% of the total cell population, cones represent 4.1%, and bipolar cells, amacrine cells, Muller cells, and astrocytes represent 9.3%, 1.4%, 5.0%, and 1.9%, respectively (Figure 1A; Table 1). Treatment with T3 greatly altered the expressions of the genes in the retinal cells. The profound alteration in gene expression was shown on the t-SNE plots (Figure 1B). There were 144 differentially expressed genes (DEGs) and 2 unmapped genes in all retinal cells prepared from the mice treated with T3 compared with the untreated controls (Supplementary Table S1). Figure 1C shows the scRNAseq detection of the expressions of the representative retinal cell-specific genes,e.g., _Opn1 sw_, _Opn1 mw_, and _Arr3_ for cones and _Rho_, _Pde6a_, _Pde6b_, _Gngt2_, and _Gnb1_ for rods, and the universally expressed genes, e.g., _Xist_ and _Cdkn1a_. We performed qRT-PCR to verify the scRNAgeq DEGs. The qRT-PCR data were consistent with the scRNAgeq data (Figure 1C,D). Thus, our scRNAgeq identified 13 cell clusters in the retina with rods representing 76.0% of the total cell population, and cones, bipolar cells, amacrine cells, Muller cells, and astrocytes representing 4.1%, 9.3%, 1.4%, 5.0%, and 1.9%, respectively.  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Cell Type** & **Cell Number (\%)** & **DEGs (\%)** & **Marker Genes** \\ \hline Rods & 118381 (76.0) & 51 (4.4) & Rho, Crugal, Pde6a, Ppref2, Nr2c3 \\ Cones & 6463 (4.1) & 450 (38.5) & Arr3, OPNmw, Gnat2, Pde6h \\ Ganglion cells & 888 (0.6) & 101 (8.6) & SclT4b, Neth, Crug, Nefl \\ Bipolar cells & 14531 (9.3) & 96 (8.2) & Camak2, Thymm, Timmen215, Crm6 \\ Amacrine cells & 2185 (1.4) & 10 (0.9) & Gad1, Ctlq2, Cabl1, Timmen215 \\ Müller cells & 7716 (5.0) & 179 (15.3) & Clue, Gluh, Apoe, RlRlP1, Carb1 \\ Horizontal cells & 979 (0.6) & 10 (0.9) & Llxx1, Oncentral, Oncentral, Cabl1 \\ Microglia & 808 (0.5) & 100 (8.6) & Cl2u, Timmen119, Afj, Cld163, Ape \\ Astrocytes & 2959 (1.9) & 163 (14.0) & Gfap, Ape \\ RPE cells & 138 (0.1) & 5 (0.4) & RlRlP1, Apoe, Carbpl1, Tppm1 \\ Pericytes & 184 (0.1) & 4 (0.3) & Acta2, Angr2 \\ Vascular cells & 30 (0.02) & & CD34, Adamtsg, Rgs5, Cd15 \\ Unsigned & 604 (0.4) & & \\ Total cells & 155866 (100) & & \\ \hline \hline \end{tabular}
\end{table}
Table 1: Cell types and DEGs in retinal cells after T3 treatment.  
Figure 1: scRNAgeq of mouse retinas after T3 treatment. (**A**) t-SNE plot of all libraries and percentage of each cell type in whole dataset. Cells are colored based on cell-type clusters. (**B**) t-SNE plot showing cell type distribution in retinas of control and T3-treated mice. (**C**) Violin plots from scRNAgeq showing expression profiles of representative genes in retinas of control and T3-treated mice. (**D**) qRT-PCR data showing mRNA fold changes in representative genes in retinas of control and T3-treated mice. Data are presented as means \(\pm\) SEM from 4 individual mice per group (* \(p<0.05\), **\(*p<0.01\)**, and *** \(p<0.001\) compared with their respective untreated controls).