DOI: 10.6084/m9.figshare.30485444.v1
title: Additional file 1 of Systematic discovery of subcellular RNA patterns in the gut epithelium

description: Additional files 1: Supplementary Figs. 1–9. Fig. S1: Proof of concept of APEX-seq in sIOs. a, Left: Scheme of the COX4 construct (also known as MITO-V5-APEX2) used as a bait to capture RNAs localizing inside of mitochondria. Right: Representative immunofluorescence images of COX4-V5-APEX2 expressing organoids. Scale bar 20 µm. b, qPCR results for the mitochondrial RNA, Mt-nd1. Results of ratio paired t test are indicated. Each dot represents one sample. Median indicated as the black bar. c, Correlation plot between Mitochondria samples with and without H2O2. A regression line is fit to log2 transformed counts per million (CPM) normalized counts to show the expression trend of the samples. Highly expressed mitochondrial genes are highlighted with dark blue color and labeled with their names (Additional Files 2: Table S4). d, Left: Scheme of the ACTB construct used as bait to capture RNAs localizing to the cytoplasm. Right: Representative immunofluorescence images of ACTB-V5-APEX2 expressing organoids. Scale bar 20 µm. e, GSEA results of ACTB enriched transcriptome from differential expression analysis using GO terms and normalized enrichment score (NES) (Additional Files 2: Table S5,6). Fig. S2: Quality control of APEX sequencing. a, Schematic representation of the constructs of DPP4 (apical bait) and GFP (cytoplasmic bait) attached to APEX2 machinery. b, Representative fluorescence of sIOs 2 days after lentiviral transduction. Scale bar 200 µm, upper panel and 20 µm, lower panel. c, Representative immunofluorescence images of sIOs stained with Streptavidin-A647 indicating biotinylated molecules in gray. Location of insets indicated with white Dashed boxes. DAPI in blue. Scale bar 20 µm and 5 µm for inset. d, The RNA integrity number (RIN) is shown for different samples. High RNA quality is insured both plus and minus H2O2 application. e, Dot blot with Streptavidin-A680. The biotinylated RNA is detected via staining with streptavidin. f, Representative correlation plots between two replicates for DPP4 and GFP samples. Each dot shows a gene and axes show normalized gene expression value for the respective sample. R value indicates Pearson correlation coefficient and p value shows the significance of calculated correlation. g, Principal component analysis (PCA) plot of samples that passed quality control (left) and percentage of explained variance by each principal component (right). h, Comparison with LCM-seq [2] (Additional Files 2: Table S6). Fig. S3: smFISH image analysis—spot detection. a, Spot detection in smFISH images. Each red dot is a smFISH dot. b, Example of segmentation of apical and basal region of cells shown on the Actb image. Lower row indicated insets in the upper row. c, smFISH image of Cdh13-mRNA. DAPI in blue and each transcript dot in white/black. Scale bar 20 µm. d, For gradient spot detection, the relative distance of each spot to the center line was considered for both apical and basal spots. Images on the right illustrate examples of spot detection and center line placement. The analysis on the right shows the spot distribution pattern relative to the total cell length for Apob-, Net1- and Cdh13-mRNA. Each dot indicates an individual transcript. Median indicated in the black bar (Additional Files 2: Table S9). Fig. S4: smFISH image analysis—foci detection. a, Diameter of RNA foci of indicated transcripts in µm (Additional Files 2: Table S10). The color of each bar plot represents their localization determined by APEX-seq (gray = unbiased, cyan = apical, magenta = basal). b, Representative smFISH images of starved and re-fed sIOs. DAPI in blue and each transcript dot in white/black. Scale bar 10 µm. White arrows indicate Lct granules in re-fed organoids. c, Quantification on smFISH dots normalized per area (dots/µm2): Lct-mRNA and Actb-mRNA. Unpaired t test with Welch’s correction applied (two-tailed p val *** &lt; 0.0001, others ns &gt; 0.05). d, Diameters of foci in µm. The number of RNA granules with &gt; 0.5 μm diameter is indicated for each condition. Unpaired t test with Welch’s correction applied (two-tailed p val **** &lt; 0.00001, ** &lt; 0.001, ns &gt; 0.05). Fig. S5: smFISH image analysis – perturbation on translation. a, Representative smFISH images of chemically perturbed sIOs. DAPI in blue and each transcript dot in white/black. Scale bar 10 µm. b, Diameters of foci in µm (Additional Files 2: Table S10). Unpaired t test with Welch’s correction applied (two-tailed p val *** &lt; 0.0001, ** &lt; 0.001, ns &gt; 0.05). Gray shows non polarized pattern, cyan for apical and magenta for basal. c, Quantification on smFISH dots normalized per area: Enpep-, Lct- and Actb-mRNA (Additional Files 2: Table S8). Unpaired t test with Welch’s correction applied (two-tailed p val ** &lt; 0.001, others ns &gt; 0.05). Gray shows non polarized pattern, cyan for apical and magenta for basal. Fig. S6: 3’UTRs carry important signals for RBP binding and for RNA localization. a, Quantification of GFP transcript spots in smFISH (Additional Files 2: Table S8). An unpaired t test with Welch’s correction is applied. *** p val &lt; 0.0001, ** p val &lt; 0.001. b, PCA plot of all samples from the RBP-MS data. c, Volcano plot of the RBP-MS data. The left side is RBPs enriched in the Fluc-mRNA binding fraction and the right side is Lct-3’UTR enriched RBPs (Additional Files 2: Table S11). d, qPCR data showing Snrnp70 is downregulated in the SNRNP70 shRNA applied sIOs compared to scrambled shRNA applied sIOs, while eGFP is not downregulated. Each dot represents technical replicates. Results of ratio paired t test are indicated. * p val &lt; 0.01. Black line indicates the median. Relative gene expression normalized to GAPDH (2 − ΔΔCT). e, Actb-mRNA smFISH images of the sIOs after Snrnp70 knock-down by shRNA and control sIOs (scrambled shRNA). DAPI in blue and each Actb transcript dot in white/black. Scale bar 10 µm. f, Left: Ratio spot density of apical to overall detected in smFISH for GFP transcripts. A Welch's t test is applied. * p val &lt; 0.01 (Additional Files 2: Table S8). Right: Quantification on Lct-3’UTR smFISH dot diameter. Unpaired t test with Welch’s correction applied (two-tailed p val **** &lt; 0.0001) (Additional Files 2: Table S10). g, Volcano plot of the RBP-MS data. The left side is Fluc enriched RBPs and the right side is Net1-3’UTR enriched RBPs (Additional Files 2: Table S12). Fig. S7: MERFISH image of a sIO section. a, Example of segmentation of apical and basal region using MERSCOPE Vizualizer. Transcripts detected from the 500 gene panel depicted in rainbow colors. PolyT (green) masks the whole cell contour: apical ROIs (toward the inner lumen) in light blue and basal ROIs in purple. Scale bar 100 µm. b, Normalized expression in apical and basal regions reveals apically and basally localizing RNAs in sIOs. The boxes show the quartiles of the data points with each dot representing one ROI. Paired Wilcoxon signed rank test is applied (Additional Files 2: Table S16). **** p val &lt; 0.00001, *** p val &lt; 0.0001, ** p val &lt; 0.001 and * p val &lt; 0.01. Fig. S8: MERFISH analysis of sIT section. a, Example of segmentation of apical and basal regions in (1) crypt, (2) villus bottom, (3) middle and (4) top, respectively. Numbers in image on the left indicate different regions for segmentation as well as apical (a) and basal (b) subregions. Transcripts for Ada, Nlpr6 or stem cell markers (see subfigure b) are depicted (Additional Files 2: Table S18). b, Localization of select transcripts confirms expected expression patterns. For example, stem cell markers (i.e., Olfm4, Sox9, Sox4, Igfbp4, Myc, Ung and Cd44 in black) are highly localized in crypts (image1). Nlrp6-mRNA in yellow is gradually localizing from villus bottom (image2) to villus top (image3) and Ada-mRNA is exclusively localizing in villus top in red (villus top marker, image3). c, Heatmap for top 30 apical and basal transcripts in different zones of sIT with log2 fold change. Villus top was taken as a reference region to select top-ranked genes (Additional Files 2: Table S17). d, The top 30 apical and top 30 basal transcripts of each zone of sIT with log2 fold change (Additional Files 2: Table S17). e, UpSet plot to compare different zones in sIT and sIOs and apical to basal, respectively (Additional Files 2: Table S19). Fig. S9: MERFISH analysis of lIT section. a, Example of segmentation of apical and basal regions in (1) crypt bottom and (2) crypt top, respectively. Crypt top and crypt bottom markers (see subfigure b) are indicated in insets. b, Stem cell marker transcripts (for example, Sox9, Mki67, and Cd44 in light gray) are localized in the crypt bottom (image1). Crypt top marker transcripts are in orange (e.g., Hnf4a, Erbb3, Irf1 and Ceacam1) (image2) (Additional Files 2: Table S20). c, Heatmap for top 30 apical and basal transcripts in different zones of lIT with log2 fold change (Additional Files 2: Table S21). d, Comparison between sIT and lIT by the top 10 apical and basal heatmap of log2 fold change (Additional Files 2: Table S18, 21). The genes were selected and aligned based on sIT’s expression profile. Colored dots indicate GO term categories. Blue dot: nutrient sensing and digesting, green dot: apical membrane receptor and receptor binding, purple dot: basal membrane receptor and receptor binding, yellow dot: pathogen defense, red dot: junctional and cytoskeletal category and gray dot: non-categorized. e, UpSet plot to compare between different zones in sIT and lIT and apical to basal, respectively.