Journal: Nature Biotechnology

Article Title: Spatial multimodal analysis of transcriptomes and metabolomes in tissues

doi: 10.1038/s41587-023-01937-y

Figure Lengend Snippet: a , The SMA workflow and quality control design—nonembedded, snap-frozen samples are sectioned and thaw-mounted onto noncharged, barcoded Visium Gene Expression arrays. Tissue sections are then sprayed with MALDI matrices and MSI is performed. This is followed by H&E staining and imaging with bright field microscopy. Finally, sections are processed for SRT. We also designed the following three types of control samples: (1) MSI—samples processed with standard MALDI-MSI protocol on ITO conductive slides; (2) VISIUM—samples processed with standard Visium protocol on all four capture areas of a Visium Gene Expression array and (3) V-iCTRL—samples processed with Visium protocol, but MALDI-MSI was performed on other capture areas of a Visium Gene Expression array. b , Pairwise gene-to-gene and molecule-to-molecule correlations across biological replicates. Samples are named with short identifiers that reflect the technical conditions under which the sample was analyzed: MSI, stand-alone MALDI-MSI; SMA, SMA protocol; VISIUM, stand-alone Visium. Additional acronyms indicate the matrix used in the SMA protocol (FMP-10, DHB and 9-AA), the sample (m1, m3 or m4) and the serial number of the tissue section (one to nine for each section placed on either ITO or Visium slides). c , UMAP of SMA ST spots colored by sections (left), MALDI matrices (middle) and clusters (right). d , Top three marker genes with highest average log 2 fold change for each spatial cluster across biological replicates. e , Spatial plot of mouse brain tissue sections (striatal level, 0.49 mm from bregma) that illustrates clusters of transcripts for samples sprayed with three different MALDI matrices (FMP-10, 9-AA and DHB) and one sample processed with the stand-alone Visium protocol.

Article Snippet: Visium Spatial Gene Expression and Tissue Optimization slides, with the exception of the human postmortem sample, were processed according to the corresponding latest versions of the 10X Genomics protocols (Visium Spatial Gene Expression Reagent Kits—Tissue Optimization User Guide, document CG000238 Rev E, 10X Genomics, (February 2022); Visium Spatial Gene Expression Reagent Kits—User Guide, document CG000239 Rev F, 10X Genomics, (January 2022) and Methanol Fixation, H&E Staining and Imaging for Visium Spatial Protocols, document CG000160 Rev C, 10X Genomics), without any modification.

Techniques: Control, Gene Expression, Staining, Imaging, Microscopy, Marker

Journal: Nature Biotechnology

Article Title: Spatial multimodal analysis of transcriptomes and metabolomes in tissues

doi: 10.1038/s41587-023-01937-y

Figure Lengend Snippet: (a) Eight mouse brain tissue sections from the striatal level of the same animal (n = 8) were mounted onto a Visium Tissue Optimization slide and sprayed with four different MALDI matrices (DHB, norharmane (analyzed in both positive and negative mode, shown as Nor+ and Nor-), 9-AA and FMP-10). Areas delimited by red lines: regions of interest imaged with MALDI-MSI. Scalebars: 1 mm. (b) Representative MSI results from: i) m/z 426.36, C18:1 L-Carnitine (DHB); ii) m/z 857.52, PI(36:4) (Nor-); iii) m/z 788.62 PC(36:1) (Nor+); iv,v) m/z 303.24, arachidonic acid (9-AA); vi) m/z 371.17, GABA (FMP-10). Nor+ and Nor-: Norharmane analyzed in positive and negative mode, respectively. Scalebars: 1 mm, except iv and v where it is 2 mm. (c) Fluorescence microscopy images of mRNA footprint captured with polydT probes after MALDI-MSI. Colored lines (i, iv, vi, viii, x, xii) demarcate areas imaged with MALDI-MSI, while gray lines (ii, iii, v, vii, ix, xi, xiii, xiv) demarcate areas not imaged with MALDI-MSI and used as controls. Scalebars: 1 mm. (d) Fluorescence intensity of tissue areas imaged or not with MALDI-MSI. The upper and lower limit of the box represent the +1 and −1 standard deviation from the mean, the horizontal line inside the box represents the mean fluorescence intensity, and the upper and lower limits of the whiskers represent the maximum and minimum fluorescence intensity values. The results shown in panels (A-C) belong to eight consecutive tissue sections from n = 1 biologically independent sample examined over one independent experiment (all the sections were placed on one Visium Tissue Optimization array). The areas in square pixels over which the statistics is derived are the following: i = 768047, ii=355349, iii=843707, iv=866085, v = 578711, vi=805789, vii=562179, viii=846042, ix=317398, x = 843416, xi=611982, xii=779667, xiii=727089, xiv=751797. (e) A mouse brain tissue sections (n = 1) from the hippocampus level was mounted onto an ITO slide and sprayed FMP-10. The area delimited by a red line demarcates the region of interest imaged with MALDI-MSI. (f) Targeted In Situ Sequencing data demonstrate similar rolling circle product (RCP) density generated from MALDI-MSI processed region (upper right panel) and non-processed region (lower right panel) for demarcated regions of interest in the mouse coronal section (n = 1). Targeted ISS simultaneously probed for housekeeping gene, Gapdh labeled in Magenta (Cy5), and a panel of five control genes - Foxj1, Plp1, Lamp5, Rorb and Kcnip2 that are labeled in Cyan (AF750). (g) Mean Cy5 and AF750 fluorescence intensity of rolling circle products in tissue areas imaged or not with MALDI-MSI.The results shown in panels (E-G) belong to one tissue section from n = 1 biologically independent sample examined over one independent experiment. The number of RCPs detected in the MALDI-MSI processed region in AF750 and Cy5 and the number of RCPs detected in the non-processed region in AF750 and Cy5 respectively, which the statistics is derived from, are the following: n = 3830,n = 18231, n = 3051,n = 18193. The lower and upper hinges of the boxplot correspond to the first and third quartiles (the 25th and 75th percentiles), the central white dot corresponds to the median, the upper and lower whiskers extend from the hinge to the maximum or minimum respectively.

Article Snippet: Visium Spatial Gene Expression and Tissue Optimization slides, with the exception of the human postmortem sample, were processed according to the corresponding latest versions of the 10X Genomics protocols (Visium Spatial Gene Expression Reagent Kits—Tissue Optimization User Guide, document CG000238 Rev E, 10X Genomics, (February 2022); Visium Spatial Gene Expression Reagent Kits—User Guide, document CG000239 Rev F, 10X Genomics, (January 2022) and Methanol Fixation, H&E Staining and Imaging for Visium Spatial Protocols, document CG000160 Rev C, 10X Genomics), without any modification.

Techniques: Fluorescence, Microscopy, Standard Deviation, Derivative Assay, In Situ, Sequencing, Generated, Labeling, Control

Journal: Nature Biotechnology

Article Title: Spatial multimodal analysis of transcriptomes and metabolomes in tissues

doi: 10.1038/s41587-023-01937-y

Figure Lengend Snippet: Violin plots and box plots illustrating the number of unique genes per spot (a) and the number of unique molecular identifiers (UMIs) per spot (b) across biological conditions of the mouse striatum data (n = 9). The numbers of spots per section from which the statistics is derived are the same for the corresponding sections in panels A and B, and are the following: V-iCTRL.FMP10.mPD3.8 = 3017, V-iCTRL.nM.mPD3.3 = 3163, SMA.9AA.mPD3.4 = 2913, SMA.DHB.mPD3.1 = 2856, SMA.DHB.mPD3.2 = 3002, SMA.FMP10.mPD1.5 = 2675, SMA.FMP10.mPD3.6 = 3120, SMA.FMP10.mPD4.7 = 2918, VISIUM.mPD3.9 = 3116. n = 9 sections examined over 3 biologically independent samples. Violin plots and box plots illustrating the number of unique genes per spot (c) and the number of unique molecular identifiers (UMIs) per spot (d) of the human striatum data (n = 1). The human sample H&E was used as a legend to indicate the four capture areas A-D. The numbers of spots per capture area from which the statistics is derived are the same for corresponding sections in panels C and D and are the following: A = 4770, B = 4875, C = 4740, D = 4387. n = 4 capture areas examined over 1 biologically independent sample. For all boxplots presented in (A-D) the lower and upper hinges of the boxplot correspond to the first and third quartiles (the 25th and 75th percentiles), the central line corresponds to the median, the upper and lower whiskers extend from the hinge to the largest or smallest value respectively no further than 1.5 times the inter-quartile range, data beyond the end of the whiskers are plotted individually as black dots. On the right, spatial featureplot representing the number of genes per spot and the number of UMIs per spot of a representative capture area (that is, capture area A). (e) Sequencing metrics: i) Gene body coverage plot illustrating the sequencing coverage at different percentiles of gene body for all the genes in the quality control dataset; ii) sequencing saturation as a function of mean reads per spot; iii) median genes per spot as a function of mean reads per spot. (f) RNA integrity plots of mouse and human post-mortem samples.

Article Snippet: Visium Spatial Gene Expression and Tissue Optimization slides, with the exception of the human postmortem sample, were processed according to the corresponding latest versions of the 10X Genomics protocols (Visium Spatial Gene Expression Reagent Kits—Tissue Optimization User Guide, document CG000238 Rev E, 10X Genomics, (February 2022); Visium Spatial Gene Expression Reagent Kits—User Guide, document CG000239 Rev F, 10X Genomics, (January 2022) and Methanol Fixation, H&E Staining and Imaging for Visium Spatial Protocols, document CG000160 Rev C, 10X Genomics), without any modification.

Techniques: Derivative Assay, Sequencing, Control

Journal: Nature Biotechnology

Article Title: Spatial multimodal analysis of transcriptomes and metabolomes in tissues

doi: 10.1038/s41587-023-01937-y

Figure Lengend Snippet: (a) Scatterplots of log 10 gene counts of SMA-SRT data vs. stand-alone Visium data. The red line highlights a 1-to-1 relationship, whereas the dashed green and blue lines highlight a log 10 0.5 or −0.5 relationship. (b) Stacked barplot illustrating the percentage of genes with log 10 higher, lower or within the log 10 fold change range −0.5-0.5. The percentages inside the gray bars illustrate the percentages of peaks with absolute log 10 below 0.5.

Article Snippet: Visium Spatial Gene Expression and Tissue Optimization slides, with the exception of the human postmortem sample, were processed according to the corresponding latest versions of the 10X Genomics protocols (Visium Spatial Gene Expression Reagent Kits—Tissue Optimization User Guide, document CG000238 Rev E, 10X Genomics, (February 2022); Visium Spatial Gene Expression Reagent Kits—User Guide, document CG000239 Rev F, 10X Genomics, (January 2022) and Methanol Fixation, H&E Staining and Imaging for Visium Spatial Protocols, document CG000160 Rev C, 10X Genomics), without any modification.

Techniques:

Journal: Journal of Cardiovascular Development and Disease

Article Title: Characterizing Neonatal Heart Maturation, Regeneration, and Scar Resolution Using Spatial Transcriptomics

doi: 10.3390/jcdd9010001

Figure Lengend Snippet: Spatially and temporally resolved transcriptional map of the regenerating neonatal mouse heart. ( A ) Schematic representation of the experimental timeline of apical resection (R) and tissue harvest for spatial transcriptomics. ( B ) 10X Visium spatial transcriptomics experimental strategy. A single representative heart was evaluated by spatial transcriptomics for each timepoint on a single slide. ( C ) Hematoxylin & Eosin stained sections used for spatial transcriptomics. ( D ) Loupe Browser was used to assign regional posts an anatomical identity, indicated by color. ( E – G ) Rank ordering of differentially expressed genes at timepoints and comparison indicated in the graph reveals the most-enriched genes that define the ( E ) ventricles versus atria; ( F ) left versus right atrium; or ( G ) left versus right ventricle. Scale bar = 1 mm.

Article Snippet: Cryosections (12 µm) were cut and placed carefully onto the specified capture area (containing the barcoded probes) on the 10X Visium Spatial Gene Expression Slide (10X Genomics, Pleasanton, CA, USA).

Techniques: Staining