Life Sciences Reporting Summary

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1 Life Sciences Reporting Summary Corresponding author(s): Christer Betsholtz Initial submission Revised version Final submission Nature Research wishes to improve the reproducibility of the work that we publish. This form is intended for publication with all accepted life science papers and provides structure for consistency and transparency in reporting. Every life science submission will use this form; some list items might not apply to an individual manuscript, but all fields must be completed for clarity. For further information on the points included in this form, see Reporting Life Sciences Research. For further information on Nature Research policies, including our data availability policy, see Authors & Referees and the Editorial Policy Checklist. Experimental design 1. Sample size Describe how sample size was determined. 2. Data exclusions Describe any data exclusions. 3. Replication Describe whether the experimental findings were reliably reproduced. 4. Randomization Describe how samples/organisms/participants were allocated into experimental groups. 5. Blinding Describe whether the investigators were blinded to group allocation during data collection and/or analysis. In this study, we aimed to sequence as many vascular single cells as possible and we achieved 3186 brain vascular cells and 1504 lung vascular cells. To our knowledge, this is the largest study ever performed in the vascular research community. No statistical methods were used to predetermine sample size. We have used the following pre-established cell exclusion criteria: cells with total aligned reads less than 100,000 were removed, and also outlier cells with maximum spearman s correlation with other cells less than 0.3 were filtered out. The gene expression profiles we reported are observed from dozens to hundreds of individual cells. All experimental findings were reliably reproduced Mice were selected for primary brain/lung cell isolation based on their genotype (i.e. fluorescent reporter expression) and age (10-19 weeks old, adult). All other criteria were not considered and as such, randomized. Selection of a specific mouse with the appropriate genotype was completely random. Clustering of the single cell data was performed using the BackSPIN algorithm, which is working in a completely unbiased fashion Note: all studies involving animals and/or human research participants must disclose whether blinding and randomization were used. 1

2 6. Statistical parameters n/a For all figures and tables that use statistical methods, confirm that the following items are present in relevant figure legends (or in the Methods section if additional space is needed). Confirmed Software The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement (animals, litters, cultures, etc.) A description of how samples were collected, noting whether measurements were taken from distinct samples or whether the same sample was measured repeatedly A statement indicating how many times each experiment was replicated The statistical test(s) used and whether they are one- or two-sided (note: only common tests should be described solely by name; more complex techniques should be described in the Methods section) A description of any assumptions or corrections, such as an adjustment for multiple comparisons The test results (e.g. P values) given as exact values whenever possible and with confidence intervals noted A clear description of statistics including central tendency (e.g. median, mean) and variation (e.g. standard deviation, interquartile range) Clearly defined error bars Policy information about availability of computer code 7. Software Describe the software used to analyze the data in this study. See the web collection on statistics for biologists for further resources and guidance. For analyse the data in this study, we used all academical softwares. For clustering the cells, we used BackSPIN software ( BackSPIN). To compare the gene expression difference between different clusters, we used R edger software (R version 3.3.2, edger version ). The 2D tsne visualization was performed using R tsne package (version 0.1-3). The heat map visualization was performed using R pheatmap packages (version 1.0.8). FACS plots were analyzed with FlowJo v10.1 and FACSDiva v Image acquisition and adjustment was done with Leica Application Suite , Fiji v1.51s and PhotoShop CC 2015, while the former two softwares were also used for quantifications. The GO analysis was performed using GOstat packages (version ) in R software, with the associated annotation packages GO.db (version 3.4.0) and org.mm.eg.db (version 3.4.0). For manuscripts utilizing custom algorithms or software that are central to the paper but not yet described in the published literature, software must be made available to editors and reviewers upon request. We strongly encourage code deposition in a community repository (e.g. GitHub). Nature Methods guidance for providing algorithms and software for publication provides further information on this topic. Materials and reagents Policy information about availability of materials 8. Materials availability Indicate whether there are restrictions on availability of unique materials or if these materials are only available for distribution by a for-profit company. No unique materials were used 2

3 9. Antibodies Describe the antibodies used and how they were validated for use in the system under study (i.e. assay and species). Antibodies were used in this study for immunofluorescence only, as validation of the transcriptomic changes we found in the single cell database. The study does not include any claims based on antibody staining only. Specificity of primary antibodies was verified in two ways: 1) Negative controls were performed using only secondary antibody. 2) Antibody staining was verfified to occur only in the correct anatomical location and cell type (based on single cell transcriptomics both from our own database and the Zeisel et al (Science, 2015) database (PMID: DOI: /science.aaa1934). To assess this, we have used the following reporter mice to visualize components of the brain vasculature: - All endothelial specific antibodies wereroutinely used in conjunction with each other and the endothelial specific Cldn5-GFP reporter mouse: CD31, Glut1, ColIV, TFRC, MFSD2a, ERG, VCAM1, Podocalyxin, VWF, AQP4 (astrocyte end-feet localized on the outer layer of vessels) - All mural cell specific antibodies were routinely used in conjunction with each other and the mural cell specific PDGFRb-GFP reporter mouse: CD13, Acta2-Cy3, Acta2-Alexa fluor 647, Acta2-FITC, PDGFRb, TAGLN, CNN1, DES - All perivascular fibroblast-like cell specific antibodies were used in conjunction with each other and the PDGFRa-H2BGFP reporter mouse: LAMA1, LUM, CD13 - GFAP staining (Z0334) was in agreement with the known morphology of astrocytes. Specific use of all antibodies is detailed below: - PECAM1 (CD31), dilution 1:250 R&D Systems cat: AF3628, lot not available as this antibody is heavily used with several lot numbers simultaneously. We have seen no variation between lot numbers in more than 5 years of use. - CD13 (anpep), dilution 1:100, Bio-Rad, cat: MCA2183EL, lot: 1113, clone R GLUT1, dilution 1:200, Merck Millipore, cat: , lot: ColIV, dilution 1:100, Bio-Rad, cat: , lot: ACTA2-Cy3, dilution 1:200, Sigma, cat: C6198, lot: 037M4783V, clone 1A4. - ACTA2-Alexa Fluor 647, dilution 1:200, Santa Cruz, cat: sc-32251, lot: E1916, clone 1A4. - ACTA2-FITC, dilution 1:200, Sigma, cat: F3777, lot: 105M4838V, 027M4784V, clone 1A4 - TFRC, dilution 1:200, Novus Biologicals; Thermo Fischer Scientific, cat: NB ; , lot: 1607, RB232679, clone H LAMA1, dilution 1:800 was a kind gift from Prof. Lydia Sorokin. Specificity of the antibody has been assessed previously on a KO mouse for LAMA1 in Edwards et al, JBC 2010: PMID: , DOI: /jbc.M PDGFRb, dilution 1:100, ebioscience, cat: , lot: E , clone APB5 - TAGLN (SM22), dilution 1:100, Abcam, cat: ab14106, lot: GR CNN1, dilution 1:100, Abcam, cat: ab46794, lot: GR AQP4, dilution 1:100, Millipore. cat: ab2218, lot: MFSD2a, dilution 1:100 was a kind gift from Prof. David Silver. Specificity of the antibody has been assessed previously on a KO mouse for MFSD2a in Nguyen et al, Nature 2014: PMID: DOI: /nature DES, dilution 1:100, Abcam, cat: ab15200, lot: GR ERG, dilution 1:200, Abcam, cat: ab92513, lot: GR VCAM1, dilution 1:100, Merck, cat: CBL1300, lot: VWF, dilution 1:100, Dako, cat: A00822, lot: SLC16a1, dilution 1:100, Acris, cat: TA321556, lot: D814AA PODXL, dilution 1:500, R&D Systems, cat: AF1556, lot: JPC , JPC LUM, dilution 1:100, Abcam, cat: ab98067, lot: GR GFAP, dilution 1:100, Dako, cat: Z0334, lot: The information above can also be found in Supplementary Table 4. For all antibodies (except LAMA1 and MFSD2a), additional information on specificity and species cross-reactivity, with links to key publications can be found on the manufacturer's website. 3

4 10. Eukaryotic cell lines a. State the source of each eukaryotic cell line used. No eukaryotic cell lines were used b. Describe the method of cell line authentication used. No eukaryotic cell lines were used c. Report whether the cell lines were tested for mycoplasma contamination. d. If any of the cell lines used are listed in the database of commonly misidentified cell lines maintained by ICLAC, provide a scientific rationale for their use. Animals and human research participants No eukaryotic cell lines were used No commonly misidentified cell lines were used Policy information about studies involving animals; when reporting animal research, follow the ARRIVE guidelines 11. Description of research animals Provide details on animals and/or animal-derived materials used in the study. Policy information about studies involving human research participants 12. Description of human research participants Describe the covariate-relevant population characteristics of the human research participants. The following transgenic mouse strains were used: C57Bl6 (The Jackson laboratory, C57BL6/J), Cspg4-dsRed (The Jackson laboratory, Tg(Cspg4-dsRed.T1)1Akik/J, Pdgfrb-eGFP (Gensat.org. Tg(Pdgfrb-eGFP)JN169Gsat/Mmucd), Pdgfra-H2B-eGFP (Pdgfratm11(EGFP)Sor, gift from Prof. Philippe Soriano), Cldn5-eGFP (Tg(Cldn5- GFP)Cbet/U, described previously), SM22-Cre (Tg(Tagln-cre)1Her/J and R26-stoptdTomato (B6;129S6-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze). All animals were backcrossed on a C57BL6/J genetic background. Adult mice of either sex, with age ranging between 11 and 19 weeks were used for all experiments. The study did not involve human research participants 4

5 Flow Cytometry Reporting Summary Form fields will expand as needed. Please do not leave fields blank. Data presentation For all flow cytometry data, confirm that: 1. The axis labels state the marker and fluorochrome used (e.g. CD4-FITC). Corresponding author(s): Christer Betsholtz Initial submission Revised version Final submission 2. The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers). 3. All plots are contour plots with outliers or pseudocolor plots. 4. A numerical value for number of cells or percentage (with statistics) is provided. Methodological details 5. Describe the sample preparation. Detailed description of sample preparation can be found in Methods section, with links to the relevant Protocols Exchange submissions. 6. Identify the instrument used for data collection. Becton Dickinson FACSAria III 7. Describe the software used to collect and analyze the flow cytometry data. 8. Describe the abundance of the relevant cell populations within post-sort fractions. FlowJo v10.1r1 Purity of samples is assessed from the single cell transcriptomes as described 9. Describe the gating strategy used. Cells were selected using forward scatter area/side scatter area (FSC-A/ SSC-A) with a very large margin for possible cell sizes and complexities, to avoid any bias in cell selection. Next, fluorescent events were selected based on the parent FSC-A/SSC-A gate. GFP was excited with a 488 nm laser, and emission was detected through a 530/30 filter, while dsred or TdTomato was exited with a 561 laser and emission detected through a 582/15 filter. A C57Bl6 mouse was used as a negative control to set the threshold for fluorescent event selection. A figure describing the gating strategy is shown in Extended Data Figure 1a Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information. nature research flow cytometry reporting summary June