Additional file 1 of Systematic discovery of subcellular RNA patterns in the gut epithelium

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 *** < 0.0001, others ns > 0.05). d, Diameters of foci in µm. The number of RNA granules with > 0.5 μm diameter is indicated for each condition. Unpaired t test with Welch’s correction applied (two-tailed p val **** < 0.00001, ** < 0.001, ns > 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 *** < 0.0001, ** < 0.001, ns > 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 ** < 0.001, others ns > 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 < 0.0001, ** p val < 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 < 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 < 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 **** < 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 < 0.00001, *** p val < 0.0001, ** p val < 0.001 and * p val < 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.

附加文件1：补充图S1至S9。
图S1：小肠类器官（small intestinal organoids, sIOs）中APEX-seq的概念验证。
a, 左图：COX4构建体（亦称MITO-V5-APEX2）的设计方案，该构建体用作捕获定位于线粒体内部RNA的诱饵。右图：表达COX4-V5-APEX2的类器官代表性免疫荧光图像。标尺为20 µm。
b, 线粒体RNA Mt-nd1的定量聚合酶链反应（qPCR）结果。标注了配对t检验的分析结果。每个圆点代表一个样本，黑色横线表示中位数。
c, 有无H₂O₂处理的线粒体样本间的相关性散点图。以log₂转换后的每百万计数（CPM）标准化计数拟合回归线，以展示样本的表达趋势。高表达的线粒体基因以深蓝色高亮并标注基因名称（附加文件2：表S4）。
d, 左图：ACTB构建体的设计方案，该构建体用作捕获定位于细胞质RNA的诱饵。右图：表达ACTB-V5-APEX2的类器官代表性免疫荧光图像。标尺为20 µm。
e, 基于基因本体（GO）术语的差异表达分析结果，展示ACTB富集转录组的基因集富集分析（GSEA）结果及标准化富集得分（NES）（附加文件2：表S5、S6）。

图S2：APEX测序的质量控制。
a, 连接于APEX2系统的DPP4（顶侧诱饵）及GFP（细胞质诱饵）构建体示意图。
b, 慢病毒转导2天后的sIOs代表性荧光图像。上图标尺为200 µm，下图标尺为20 µm。
c, 用链霉亲和素-A647染色的sIOs代表性免疫荧光图像，生物素化分子以灰色显示。白色虚线框标注了放大区域的位置。DAPI以蓝色显示。标尺为20 µm，放大区域标尺为5 µm。
d, 不同样本的RNA完整性数（RIN）结果，证明H₂O₂处理组与对照组均具有较高的RNA质量。
e, 链霉亲和素-A680斑点印迹结果，通过链霉亲和素染色检测生物素化RNA。
f, DPP4及GFP样本的两个生物学重复间的代表性相关性散点图。每个圆点代表一个基因，坐标轴分别为对应样本的标准化基因表达值。R值表示皮尔逊相关系数，p值为相关性检验的显著性结果。
g, 通过质量控制的样本的主成分分析（PCA）散点图（左图），及各主成分解释的方差百分比（右图）。
h, 与激光捕获显微切割测序（LCM-seq）[2]的比较结果（附加文件2：表S6）。

图S3：单分子荧光原位杂交（single-molecule fluorescence in situ hybridization, smFISH）图像分析——斑点检测。
a, smFISH图像中的斑点检测，每个红色圆点代表一个smFISH斑点。
b, Actb图像中细胞顶侧与基侧区域的分割示例，下方行对应上方行的放大区域。
c, Cdh13信使RNA（mRNA）的smFISH图像。DAPI以蓝色显示，每个转录本斑点以白色/黑色显示。标尺为20 µm。
d, 对于梯度斑点检测，需考虑每个斑点相对于中心线的相对距离，分别针对顶侧及基侧斑点。右侧图像展示了斑点检测及中心线放置的示例，右侧分析图展示了Apob、Net1及Cdh13 mRNA相对于细胞总长度的斑点分布模式。每个圆点代表单个转录本，黑色横线表示中位数（附加文件2：表S9）。

图S4：smFISH图像分析——聚集焦点检测。
a, 所示转录本的RNA聚集焦点直径，单位为µm（附加文件2：表S10）。每个柱状图的颜色代表通过APEX-seq确定的定位类型：灰色=无偏向性，青色=顶侧定位，品红色=基侧定位。
b, 饥饿及复喂养sIOs的代表性smFISH图像。DAPI以蓝色显示，每个转录本斑点以白色/黑色显示。标尺为10 µm。白色箭头指示复喂养类器官中的Lct颗粒。
c, 单位面积smFISH斑点数量的定量结果（斑点数/µm²）：Lct-mRNA及Actb-mRNA。采用Welch校正的非配对t检验（双侧检验：*** p<0.0001，其余无统计学差异ns>0.05）。
d, RNA聚集焦点的直径分布，标注了各条件下直径>0.5 µm的RNA颗粒数量。采用Welch校正的非配对t检验（双侧检验：**** p<0.00001，** p<0.001，其余ns>0.05）。

图S5：smFISH图像分析——翻译扰动实验。
a, 化学扰动处理的sIOs的代表性smFISH图像。DAPI以蓝色显示，每个转录本斑点以白色/黑色显示。标尺为10 µm。
b, RNA聚集焦点的直径分布（附加文件2：表S10）。采用Welch校正的非配对t检验（双侧检验：*** p<0.0001，** p<0.001，其余ns>0.05）。颜色说明：灰色=无偏向性定位，青色=顶侧定位，品红色=基侧定位。
c, 单位面积smFISH斑点数量的定量结果：Enpep、Lct及Actb mRNA（附加文件2：表S8）。采用Welch校正的非配对t检验（双侧检验：** p<0.001，其余ns>0.05）。颜色说明：灰色=无偏向性定位，青色=顶侧定位，品红色=基侧定位。

图S6：3'非翻译区（3' untranslated region, 3'UTR）携带RNA结合蛋白（RNA-binding protein, RBP）结合及RNA定位的关键信号序列。
a, smFISH中GFP转录本斑点数量的定量结果（附加文件2：表S8）。采用Welch校正的非配对t检验，*** p<0.0001，** p<0.001。
b, 所有RBP-MS样本的PCA散点图。
c, RBP-MS数据的火山图。左侧为富集于Fluc-mRNA结合组分的RNA结合蛋白，右侧为富集于Lct-3'UTR的RNA结合蛋白（附加文件2：表S11）。
d, qPCR结果显示：与阴性对照短发夹RNA（scrambled shRNA）处理的sIOs相比，Snrnp70短发夹RNA（shRNA）处理的sIOs中Snrnp70基因表达下调，而eGFP基因表达无显著变化。每个圆点代表技术重复，标注了配对t检验的分析结果。* p<0.01，黑色横线表示中位数。相对基因表达以GAPDH为内参进行标准化（采用2^(-ΔΔCT)法计算）。
e, Snrnp70基因敲低及对照（scrambled shRNA）处理的sIOs的Actb-mRNA smFISH图像。DAPI以蓝色显示，每个Actb转录本斑点以白色/黑色显示。标尺为10 µm。
f, 左图：smFISH中GFP转录本的顶侧斑点密度与总斑点密度的比值，采用Welch t检验，* p<0.01（附加文件2：表S8）。右图：Lct-3'UTR的smFISH斑点直径定量结果，采用Welch校正的非配对t检验（双侧检验：**** p<0.00001）（附加文件2：表S10）。
g, RBP-MS数据的火山图。左侧为富集于Fluc的RNA结合蛋白，右侧为富集于Net1-3'UTR的RNA结合蛋白（附加文件2：表S12）。

图S7：sIO切片的多重错误鲁棒荧光原位杂交（multiplex error-robust fluorescence in situ hybridization, MERFISH）图像。
a, 使用MERSCOPE可视化软件进行细胞顶侧与基侧区域分割的示例。500基因面板检测到的转录本以彩虹色显示。聚胸腺嘧啶（polyT，绿色）标记整个细胞轮廓：顶侧感兴趣区域（朝向内部管腔）为浅蓝色，基侧感兴趣区域为紫色。标尺为100 µm。
b, 顶侧与基侧区域的标准化表达量，揭示了sIOs中定位于顶侧及基侧的RNA。箱体图展示数据点的四分位数，每个圆点代表一个感兴趣区域。采用Wilcoxon符号秩检验（附加文件2：表S16），**** p<0.00001，*** p<0.0001，** p<0.001，* p<0.01。

图S8：小肠组织（small intestinal tissue, sIT）切片的MERFISH分析。
a, 不同区域的顶侧与基侧区域分割示例：(1)隐窝、(2)绒毛底部、(3)绒毛中部、(4)绒毛顶部。左侧图像中的数字标注了分割的不同区域，以及顶侧（a）与基侧（b）子区域。展示了Ada、Nlpr6或干细胞标志物的转录本（附加文件2：表S18）。
b, 选定转录本的定位验证了预期的表达模式：例如干细胞标志物（如Olfm4、Sox9、Sox4、Igfbp4、Myc、Ung及Cd44，黑色）高度富集于隐窝（图1）；Nlrp6 mRNA（黄色）从绒毛底部（图2）到绒毛顶部（图3）逐渐富集；Ada mRNA仅富集于绒毛顶部（红色，绒毛顶部标志物，图3）。
c, 不同sIT区域中前30个顶侧及基侧转录本的热图，以log2倍数变化展示。以绒毛顶部作为参考区域筛选排名靠前的基因（附加文件2：表S17）。
d, 不同sIT区域中前30个顶侧及前30个基侧转录本的log2倍数变化结果（附加文件2：表S17）。
e, UpSet图，分别比较sIT不同区域、sIO不同区域以及顶侧与基侧样本间的基因交集（附加文件2：表S19）。

图S9：大肠组织（large intestinal tissue, lIT）切片的MERFISH分析。
a, 不同区域的顶侧与基侧区域分割示例：(1)隐窝底部、(2)隐窝顶部。隐窝顶部及隐窝底部标志物的放大区域见插图。
b, 干细胞标志物转录本（如Sox9、Mki67及Cd44，浅灰色）富集于隐窝底部（图1）；隐窝顶部标志物转录本为橙色（如Hnf4a、Erbb3、Irf1及Ceacam1）（图2）（附加文件2：表S20）。
c, 不同lIT区域中前30个顶侧及基侧转录本的热图，以log2倍数变化展示（附加文件2：表S21）。
d, 基于sIT的表达谱筛选基因，对sIT与lIT的前10个顶侧及基侧转录本的log2倍数变化进行比较（附加文件2：表S18、S21）。彩色圆点代表基因本体（GO）术语类别：蓝色圆点=营养感知与消化，绿色圆点=顶膜受体及受体结合，紫色圆点=基膜受体及受体结合，黄色圆点=病原体防御，红色圆点=连接与细胞骨架类别，灰色圆点=未分类。
e, UpSet图，分别比较lIT不同区域、sIT与lIT间以及顶侧与基侧样本间的基因交集。