Analysis of single mRNA and sgRNA samples for mouse ES cells using TACIT, Samtools, and MACS for aggregated analysis

The median gene expression for the synthetic cell was derived from the cells of the same cell. Each chromatin region was linked to the nearest genes using Homer, and expression for all genes and all samples were then combined and split by categories of chromatin states. The box plot was for each state. Synthetic cells at the 2cell and 4cell stages were included in the analysis to eliminate the effects of non-canonical features.

In order to insert the sgRNAs targeting the promoter of each candidate totipotency-related TF, a CROP-optiVector was created. The libraries of sgRNAs were candidate, positive control and non-targeting. The supernatant with lentivirus was collected 18 h after transfection and filtered to remove cell debris. The mouse ES cells were infected (8 μg ml–1 polybrene) with various titres of lentivirus to achieve different multiplicity of infection values. After 24 h, a new culture medium with 2 gml–1 puromycin was added. Cells after transduction and selection were collected for scRNA-seq. The cells were put in a box at 4 C for 10 minutes, then preserved at an ambient temperature of 80 C. The single-cell library was done for capturing both mRNA and sgRNAs for mouse ES cells.

There were modifications to the data for single cells. Raw TACIT sequencing data were evaluated using FastQC (v.0.11.5), followed by mapping to the mouse reference genome mm10 by Bowtie2 (v.2.2.9)55. Mapped reads with MAPQ vales less than 30 were considered as multi-mapped reads and filtered out using Samtools (v.1.9). The same thing was done using Picard (v.2.2.4). For aggregated analysis, single-cell .bam files were merged with Samtools. For peak calling, MACS2 (v.2.1.1)56 with the ‘–broad’ parameter was used to call peaks for aggregated profiles of TACIT data. The raw CotacIT data were de-multiplexed using an in-house code. Sequencing data for each histone modification was performed according to the analysis pipeline as described for TACIT data.

The allvalid pairs matrix for late 2cell stage was downloaded from the GEO database. To identify interactions, we used the analyze HiC function of Homer and plotted interactions with Python.

H3K4me1, H3K4me, H3K36me and H3K27ac were integrated with genes. In brief, the cell–peak or cell–bin matrix for each histone modification was first generated using cisTopic58. The GeneActivity function of Seurat (v.4) was used to create a gene-activity score matrix based on the cell–peak or cell–bin matrix. The FindTransferDans function was used to locate the anchors between the two modalities. Many titrations were performed to obtain the highest prediction score, including using the peak or bin matrix, or the cell–bin matrix. The cells with a prediction score lower than 0.2 were removed. Histone modifications in non-Canonical broad binding regions were excluded before Seurat integration for the 2cell stage.

In order to build a random forest machine-learning model and to use synthetic cells as testing cells we used 29 of them as training cells and 10 of them as testing cells. This trained random forest model was used to estimate the ICM or TE tendency of 4cell, 8cell and morula cells.

We used the probability matrix for each state as input for clustering cells and for UMAP visualization. We used 1:5 dimensions for clustering. The average probability of a specific chromatin state in eachTSS was averaged from genomic intervals that were 2kb flanking TSS regions, and for clustering cells on the basis of annotations in allTSS. Next, we used the mean probability matrix for each chromatin state for TF–IDF normalization, SVD dimensionality reduction, cluster finding and UMAP visualization. The dimensions were used for visualization and clustering.

The model we used for learning was a 12-state model with aggregate ICM andTE profiles. We learned the probabilities of the interpolated single cells with the forward–backward model. The states were grouped into 6 categories based on the bin size. We also merged five adjacent single cells along pseudotime52.

Having obtained 155 RNA synthetic cells interpolated with six histone-modification profiles, we performed hierarchical clustering with RNA synthetic cells on the basis of multimodal histone modifications. The cell number of the stage is related to the number of clusters, which in this case are two for the 2cell stage and four for the 4cell stage. There were 90 synthetic single cells with joint profiles of 6 histones in the aggregated histone-modification profiles. To reduce effects from sequencing depth, we normalized cell numbers and non-duplicated reads before aggregating data.

In step (2), we described how H3K 27ac CoTACIT profiles with gene expression can be integrated. The H3K 27ac, H3K27me3 and H3K9me3 profiles were transferred to the linked RNA synthetic cells.

We ordered scRNA-seq cells along the developmental trajectory using Monocle3 (ref. 60) and merged five adjacent single cells along pseudotime into one RNA synthetic cell.

We called peaks for aggregated.bam files of each histone modification to calculate the genome coverage at each stage. We called peaks with parameters of no model and nolambda. Next, we binned the mm10 genome into 200-bp genomic intervals, and for each histone modification, genome coverage at a specific stage was calculated as the percentage of genome intervals that overlapped with peaks at that stage. To evaluate the coverage of single cells, the genome was first binned and histone signals were defined as covered bins. The percentage of covered bins was defined as genome coverage for each single cell.

The multiBigwigSummary function in deepTools helped us to calculate the normalized mean scores for different experiments. The Spearman correlation or Pearson correlation was calculated between replicates and plotted using the plotCorrelation function.

Structural characterization of spermocytes using blocking buffer and secondary antibody incubation for 4 h at room temperature. Embryos were collected and stained with PAT

Injected embryos were fixed with 4% paraformaldehyde (Sigma) for 10–15 min. PBST (PBS + 0.5% Triton-X) was added for 20 min at room temperature to permeabilize the embryos and the samples were subsequently incubated with blocking buffer (PBS + 0.1% Tween-20 + 5% NDS) for 4 h at 4 °C. After blocking, embryos were incubated with SOX2 (Active motif) and CDX2 (BioGenex), diluted in blocking buffer, at 4 °C overnight. Samples were then washed with PBS 3 times and incubated with secondary antibodies (Invitrogen), diluted in blocking buffer, for 2–4 h at room temperature. After being acclimatized with 600 nM DAPI solution for 5 minutes at room temperature, blastocysts were ready to be visualized. The images were taken using a confocal microscope.

Next, cells were incubated with specific antibody in 100 μl antibody buffer (20 mM HEPES pH 7.5, 150 mM 0.1 mM spermidine, 0.05% digitonin, 1 mMBSA–PBS, 1 cocktail, 10mM sodium butyrate and 1 mM PMSF are present at 4 C. After incubation, cells were washed twice with 180 μl Dig-wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine (Sigma), 0.01% digitonin, 0.05% TX-100, 1× cocktail, 10 mM sodium butyrate and 1 mM PMSF) and suspended with 100 μl high-salt Dig-wash buffer (20 mM HEPES pH 7.5, 300 mM There is a 0.25 mM spermidine, 0.05% digitonin, 0.05% TX-100, 10 mM sodium butyrate and 1 mM PMSF. The PAT expression, purification and assembly procedures were performed as per previously described guidelines19. Cells were rotated at 4 °C for 1 h to enable complete binding of PAT to antibodies and then washed twice with 180 μl high-salt Dig-wash buffer to remove free PAT–MEA/B. Tagmentation was reactivated by suspending cells with 10 μl cold reaction buffer (10 mM TAPS-NaOH pH 8.3, 5 mM MgCl2, 1× cocktail, 10 mM sodium butyrate and 1 mM PMSF) and incubated at 37 °C for 1 h in a PCR cycler. Adding 10 l 40 mM stopped the reaction. The cells were washed twice with 1%BSA–PBS, and put into a well of a 96-well plate with a mouth pipette under a microscope. Pre-rinsed 96 well plates have a 2 llysis buffer to prevent loss of DNA fragments. Tris-HCl pH 8.5, 0.05% SDS and 0.1 mg ml–1 proteinase K) was added to each well. For each well, samples were covered with mineral oil and held at 55 C for 15 minutes. There will be 1.0 l of 10 mM next. Adding PMSF and 0.9% of Triton X-100 to the wells made a datememe datememe. 17 l mix is finally here. KAPA HiFi HotStart DNA polymerase, 4 μl 5× KAPA High-GC buffer, 0.5 μl 10 mM dNTP mix, 0.5 μl 25 mM MgCl2 and 10.8 μl H2O) was added to each well with 0.5 μl 10 mM The Nextera i5 and i7 index primer are included in the Supplementary Table 1. A thermal cycler was used to perform the enrichment, with 1 cycle of 72 C for five minutes, 1 cycle of 95 C for three minutes, and 11 cycles of 98 C for 20 minutes, 65 C for 30 minutes. The library was purified with 1 AMPure XP beads once, and the remaining 200–1,000 bp fragments were selected with a small amount of placebos. AMPure XP beads are made from a mixture of metal and glass. The libraries were sequenced with paired-end 150-bp reads on a NovaSeq 6000 platform (Illumina).

For siRNA knockdown, isolated zygotes were microinjected with sets of three siRNAs against targets (20 μM in total) or with non-target control (NC, 20 μM in total). The following siRNAs were used: NC, UGGGACUUGCAGGCCUGAUAUTT; Nanog, CGAGAACUAUUCUUGCUUATT, CCUGAGCUAUAAGCAGGUUAATT and UGGAGUAUCCCAGCAUCCAUUTT; Zfx, GGUUCAUGAUAGUGUAGUATT, GGAUGAAGAUGGACUUGAATT and GGAGGACAACGAAAUGAAATT; Yy2, GCUGCGAGAAGAUGUUCAATT, CACCAUGUGGGACGAUGUUAATT and GACCUAUAGCAUGCUCUCAUATT; Tcf12, GUGGCAGUCAUCCUUAGUCUATT, GAUGCAAUGUCCUUCUUAATT and GGAACAAGUGGUCAACCAATT; Cebpb, GAGCGACGAGUACAAGAUGTT, CACCCUGCGGAACUUGUUCAATT and CGCCUUUAGACCCAUGGAAGUTT; Bbx, UGGGACUUGCAGGCCUGAUAUTT, CCAGUGGGAGCAAGAAGUUUATT and CUCCCUCAAUAUAGUCCUAUUTT; Smad2, GUGAUAGUGCAAUCUUUGUTT, UGGUGUUCAAUCGCAUACUAUTT and CCUUCAGUGCGAUGCUCAATT; Hbp1, CCCUACCCAAUCUGCCAUAUATT, GGCUAACAGAGUUAGCAAATT and CCAGCUAAGUUCAGAUGUATT; Cdx2, GGACAGAAGAUGAGUGGAATT, GAGAAGGAGUUUCACUUUATT and GCUUGCUGCAGACGCUCAATT; Klf6, GCUUGCUGCAGACGCUCAATT, GACCAAUAGCCUGAACUCUTT and GAUGAGUUGACCAGACACUTT; Sox15, CCUGGCAGUUACACCUCUUCTT, GAUGAAGAGAAGCGACCCUUTT and GACUCUUCCACUCCAUAUAAUTT; Med1, UAAGCUUGUGCGUCAAGUAAUTT, GGCUCUCCAAUCCUUAGAACATTand GUGGCCUAUAACACUCUAAUUTT; Elf5, GCCCUGAGAUACUACUAUAAATT, GGACCGAUCUGUUCAGCAATT and GGAGGUUAGUGUACAAAUUTT; and Hif1A, CCAUGUGACCAUGAGGAAATT, GCAGACCCAGUUACAGAAATT and GCAGGAAUUGGAACAUUAUTT. Hippobio gave the order for the siRNAs. The injected embryos were transferred to KSOMaa medium (Millipore) and droplets were covered with mineral oil (Sigma) in a Petri dish (Ibidi) and cultured in a tissue incubator (37 °C and 5% CO2) (Thermo Fisher Scientific). At the 8cell or blastocyst stage, single-embryo RNA-sq or immunofluorescence staining was performed to confirm KD or marker gene expression.

The following antibodies were used in TACIT: H3K4me 1 and H3K4me3. The donkey anti-rabbit-Alexa 484 and donkey anti-rabbit-Alexa 547 were used as secondary antibodies. Antibodies used in immunofluorescence staining included SOX2 (1:200; Active Motif, 39843, 2226414) and CDX2 (1:200, BioGenex, MU392A-UC, MU392A0516D).

The mouse cell culture was carried out at 37 degreesC with 5% CO2 and was maintained on 0.1% jello-coated plates.

Source: Genome-coverage single-cell histone modifications for embryo lineage tracing

Data handling and statistical analyses of experiments on light-dark cycles: C57BL/6 mice from the National Institute on Aging rodent colony

A mass containing several zygotes surrounded by cells was transferred to a small sample of hyaluronidase solution and then incubated at 37 C. The zona pellucida was gently removed by treating with a pre-warmed Tyrode’s acidic solution. The second bodies of the animal were removed with a glass needle.

Aged C57BL/6 mice (16–21 months old) were obtained from the National Institute on Aging rodent colony. Young C57BL/6 mice (3 months old) were obtained from Jackson Laboratories. All the experiments used male mice. All of the mice were kept on a light–dark cycle and had access to food and water. The procedures for animal care complied with the Animal Welfare Act and were approved by the Veterans Affairs Palo Alto Committee on Animal Research as well as the institutional administrative panel of laboratory animal care at Stanford University.

There were data handling and statistical analyses performed with R studio and GraphPad. The analyses were carried out using t-tests. All statistical analyses comparing measurements between three or more groups were carried out using one-way ANOVA tests with post hoc tests for multiple comparisons. The P value of 0.05 was considered significant. All experimental procedures were carried out in alternating ways to avoid temporal and technical biases. Data in Figs. 2a,b,f–j, 3e–n, 4f and 5j–l are extended data figs. 2a, 3f–i, 7a–c,h,i and 8h–k were successfully replicated in at least two independent experiments.

In fear conditioning tests, mice were trained to associate cage context and an audiovisual cue with an aversive stimulus (foot shock). The mice were exposed to 30 s of paired cue light and a 1,000-hertz tone after being placed in a cage. On day 2 (contextual fear conditioning), mice were re-exposed to the same cage context and freezing behaviour was recorded during minutes 1.5 to 6 using a FreezeScan tracking system (Cleversys). On day 4 (cued fear conditioning), mice were placed in a novel context that contained different odour, floor texture, and chamber walls and re-exposed the same cue light and tone from day 1 after 2 min of exploration. Freezing behaviour was recorded for 1.5–6 min following the cue using the FreezeScan tracking system (Cleversys).

The Y maze is made up of 3 white, opaque plastic arms that are 120 from each other. At the beginning of the trials, mice were placed in the end of 1 arm and allowed to freely explore all 3 arms for 5 min. The definition of an arm entry was that it had all four limbs in an arm. The maze was cleaned with 70% ethanol between animals and before the first animal to eliminate traces of odour. The number of arm entries and triads (a set of consecutive arm entries) were recorded. The number of triads was divided by the number of possibilities to arrive at the number of possible changes.

P value correction, bulk data normalization and differential gene expression in the M. musculus Reference Genome GRCm38 using DESeq2

AggregateExpression function was used to derive pseudobulk counts from raw counts. DESeq2 was then used to perform bulk data normalization and differential gene expression across groups using default parameters. P value correction was carried out using the Benjamini–Hochberg procedure (FDR = 0.05) for each comparison. Genes with FDR <0.05 were used for subsequent KEGG pathway enrichment and Metascape43 analysis (Supplementary Data 5).

Gene counts were obtained by aligning reads to the M. musculus reference genome GRCm38 using CellRanger software (v4.0.0) (10X Genomics). Ambient RNA was removed from each sample using SoupX (v1.6.2) and droplets containing multiple nuclei were filtered out using DoubletFinder (v2.0.4). We used Seurat (v4.1.1) to further exclude cells with fewer than 200 or more than 5,000 features and cells with more than 10% mitochondrial genes. In total, 69,250 cells remained and were used for further analysis. 3a–d

Source: Glycocalyx dysregulation impairs blood–brain barrier in ageing and disease

Microvessel dissociation of young mice and production of Alexa Fluor-conjugated Streptavidin for AAV production

Microvessels from young (3-month-old) and aged (21-month-old) mice were isolated as previously described with some modifications26,34. In brief, mice were euthanized via CO2, and brains were retrieved in PBS supplemented with 1% bovine serum albumin (BSA) and 1× cOmplete protease inhibitor cocktail (Millipore Sigma) on ice. The olfactory bulb and meningeal vessels had to be removed with blotting paper. Brains were minced using a razor blade on ice and then homogenized using a loose- fit, 7 liter container. You should dounceWheaton with 1 protease inhibitor. The 70 kDa dextran (Sigma) was added to the homogenate at a rate of 4,400g per min. Myelin and parenchymal cell layers were removed. Pelleted microvessels were deposited on a pre-wet 40-μm strainer, washed with PBS, and mechanically dissociated into single cells as previously described26. For enzymatic dissociation of brain tissue, previously published protocols were used12,13. Cells were stained with the following antibodies after they had been suspended in FACS buffer for 30 minutes. After washing cells with FACS buffer, secondary incubation with Alexa Fluor-conjugated streptavidin (1:1,000, Thermo Fisher Scientific) and secondary antibodies (1:400, Thermo Fisher Scientific) was carried out on ice for 20 min. Live cells were identified using Sytox Blue viability dye (1:1,000, Thermo Fisher Scientific, S34857). Flow cytometry analysis was performed on a BD LSRFortessa, and data were analysed using FlowJo software (TreeStar).

For production of PHP.V1-sCLDN5::EGFP and all other plasmids containing the PHP.V1 (pUCmini-iCAP-PHP.V1 was a gift from V. Gradinaru; Addgene plasmid #127847) capsid, AAV production was performed in-house utilizing a previously published protocol42. In brief, triple transfection of HEK293T cells was performed on 90–95% confluent cells in DMEM containing Glutamax supplemented with 5% FBS and non-essential amino acids. A warm medium was replaced 12 hours after thefection. The medium was collected for 72 hours. A fresh, warm medium was added as well as cells 120 h after thefection. The cells and medium were put in the freezer and then put back in the machine for 15 minutes. Supernatant was collected in a separate bottle and combined with 40% (wt/vol) Before transferring to 4C overnight, incubating PEG on ice for two hours, you can add 8% wt/vol. A buffer containing salt- active nuclease was used to raise the cell pellet to 37 C for one hour before it was moved to 4 C. The medium was 4,000g for 30 min at 4 C. After centrifugation, supernatant was bleached and discarded. The pellet was resuspended in a SAN + SAN buffer, plus the previous fraction and then put in a 37 C incubator for 30 minutes. For 15 minutes, lysate and supernatant were loaded onto an Iodixanol gradient by means of acentrifugation. Gradients were transferred to an ultracentrifuge (Beckman Coulter) using a Type 70 Ti rotor set at 350,000g for 2 h and 25 min at 18 °C. AAV particles were collected from the 40/60% interface, washed in PBS, and concentrated using an Amicon Ultra-15 (Millipore Sigma) filter device with a 100 kDa cutoff. AAV titration was performed using the AAVpro Titration Kit (for Real Time PCR) Ver.2 (Takara Bio). AAVs were injected retro-orbitally at 8 × 1011 viral genomes per mouse.

The number 26 is called the Ple. MiniPromoter was a gift from E. Simpson. A cis rAAV genome plasmid with AIV2 inverted terminal repeats was used for cloning of a sCLDN5 and EGFP reporter. To knock down C1galt1 in brain endothelial cells, de novo predictions of small interfering RNA (siRNA) guides targeting C1galt1 were generated using the DSIR algorithm39 and subsequently filtered using ‘Sensor rules’ to select for sequences with highly favourable small hairpin RNA (shRNA) features40,41. Three de novo 97-mer miR-E shRNA sequences (Supplementary Table 2) were synthesized (IDT) and inserted into pAAV-sCLDN5-EGFP using restriction enzyme cloning for in vitro evaluation. To overexpress C1GALT1 and B3GNT3 in brain endothelial cells, P2A-C1GALT1 and P2A-B3GNT3 were cloned into pAAV-sCLDN5-EGFP using restriction enzyme cloning to generate pAAV-sCLDN5-EGFP-P2A-C1GALT1 and pAAV-sCLDN5-EGFP-P2A-B3GNT3, respectively.

Cerebral microvessels were isolated for immunofluorescence imaging using the same protocol described above for flow cytometry, except, instead of dissociating microvessels, they were fixed on 40-μm strainers with 4% PFA in PBS at room temperature for 15 min with gentle rocking. Microvessels were washed with PBS and mounted on slides. Microvessels were blocked in 3% normal donkey serum (Jackson ImmunoResearch) with 0.3% Triton X-100 (Sigma) in TBS-T (1× TBS with 0.05% Tween-20) for 1 h, followed by 1 h incubation at room temperature with primary antibodies. For glyco-profiling of cerebral microvessels, the same primary antibodies, lectins, binding proteins and concentrations from our flow panel were used along with fluorescein-conjugated VVA (1:300, Vector Labs, FL-1231-2). A rabbit anti-CAV1 is one of the additional primary antibodies used. Microvessels were washed three times with TBS-T for 5min each, followed by incubation with Fluor-conjugated antibodies or streptaviadin for 1 h at room temperature. Microvessels were washed three more times and then covered with something called the Vectashield Hardset Antifade mounting medium or ProLong Gold Antifade Mountant. It was done on a confocal laser-scanning microscope. The observed single plane microvessels were quantified using ImageJ software.

bEnd.3 cells were treated with 5 nM StcE at 37 C after being plated in a 24well plate. Cells were collected from plates using enzyme-free cell dissociation buffer (Thermo Fisher Scientific, 1315014) and resuspended in 1% BSA in PBS (FACS buffer). The cells were stained with the following anti-proliferative drugs in FACS buffer for 30 min. Live cells were identified using Sytox Blue viability dye (1:1,000, Thermo Fisher Scientific, S34857). Data was analysed using FlowJo software, which was used for the Sony SH 800S sorter analysis. Only live, singlet cells were used for analysis of adhesion molecule MFI.

The mouse brain endothelial cell line bEnd.3 was kept in a humidified incubator supplemented with 10% fetal bovine serum and 1% penicillin/streeptomycin. bEnd.3 cells were grown in 6well plates and treated with 5 nM StcE for 16 h at 37 C. Cells were lysed and collected into RNAse-free Eppendorf tubes for total RNA extraction using the RNeasy Plus Micro kit (Qiagen, 74034). RNA quantity and quality were assessed by an Agilent 2100 Bioanalyzer (Agilent Technologies). All samples passed the high quality control threshold and were ready for library preparation by Novogene. Libraries were sequenced on the NovaSeq 6000 (paired-end, 2× 150 bp depth). The M. musculus reference genome was aligned withrimmed reads. The analysis and visualization of differential genes were done using DESeq2. GO biological pathway enrichment analysis uses genes with a Padj 0.05.

Previously published ageing and neurodegenerative disease RNA-seq datasets demonstrating robust brain endothelial cell enrichment were chosen for glycosylation-related gene analysis12,26,27. We filtered for glycosylation-related genes based on KEGG (Kyoto Encyclopedia of Genes and Genomes) listed glycosylation enzymes and related proteins. Most glycoproteins were excluded due to the enormous variety of members in this family which possess biological functions not directly relevant to glycosylation. Significantly upregulated and downregulated glycosylation-related genes in each dataset were used for Reactome pathway analysis with Padj < 0.05 set as the threshold for significant enrichment.

The LC–MS/MS analysis was performed on a Q Ex Active HF-X with an UltiMate 3000RsseC system. There was a 75-m capillary column packed with 40 cm of ReproSil-PUR 120 C18-AQ 1.9 m resin that was in-house. Chromatographic separation was achieved using a flow rate of 300 nl min−1 with the following 120 min gradient: 96% A + 4% B for 18 min, 70% A + 30% B for 72 min, 60% A + 40% B for 15 min, and 4% A + 96% B for 15 min, where solvent A was 0.1 % formic acid in HPLC-grade water (Fisher) and solvent B was 0.1% formic acid in HPLC-grade acetonitrile (Fisher). Full MS scans were acquired at a resolution of 60,000, with an automatic gain control (AGC) target of 3 × 106, maximum injection time (IT) of 20 ms, and scan range 300–1,650 m/z in a data-dependent mode. With the parameters included, the scans were able to be obtained with a resolution of 15,000, AGC target of 1 105, loop count 15, TopN 15, and isolation window 1.4 m/z. There were several matches made against the Mus musculus reference proteome database. Variable modifications were specified for methionine oxidation and N-terminal acetylation. The searches used for thecursor ion and product ion mass tolerances were based on 20 ppm. The unique and razorpeptides were used for analysis. There was a 1% false discover rate for the results at the peptides andProtein levels. MaxLFQ37 was used to calculate the minimum ratio count, and the ratio count was set to 1. For quantitative comparative analysis, protein intensity values were log2-transformed, and missing values were imputed from a normal distribution with width 0.3 and downshift value of 1.8 using Perseus. The Benjamini–Hochberg FDR was used to perform the principal component analysis in Perseus. DAVID38 was used to perform GO term enrichments.

Plasma cytokine measurement was performed using the Luminex assay at the Human Immune Monitoring Center at Stanford University. The procarta kit was used according to the instructions from the manufacturer. The quality control samples included a standard curve and a singlet on a 96-well plate. The control beads were added to the wells to assess nonspecific binding.

The production of all StcE proteins was described. For all applications, proteins were run through Pierce high-capacity endotoxin removal columns (Thermo Fisher Scientific) at least seven times following manufacturer’s instructions. The levels of endotoxin were tested using the kit from InvivoGen. Mice were injected retro-orbitally with 0.25 mg kg−1 StcE once a day for 2 days before perfusion with ice-cold PBS. Cerebral bleeding was seen by the eye post-perfusion. Hemibrains and peripheral organs were cut into 5m-thick sections with slides for H&E staining. Sections were deparaffinized with xylene and stained with Richard Allan haematylin after they were hydrated in a series of graded alcohols. Sections were then dehydrated (10 dips in 95% ethanol followed by 2× 1 min in 100% ethanol), cleared in xylene (3 times, 1 min), and coverslipped prior to imaging on a wide-field microscope (Zeiss AxioImager).

The Mice were injected at 0.25 g1 body weight with a retro-orbital solution of Sulfo-NHS-biotin. The tracer was allowed to circulate for 5 min before perfusion with PBS. The brains were sectioned into 40m slices after they were post-fixed in 4% PFC. Sections were blockaded with CD31 and the appropriate secondary antibody, as was described earlier. Images were taken on a confocal laser-scanning microscope (Zeiss LSM880) and analysed using ImageJ software. The permeability index of vessels was determined by dividing the area occupied by tracer by the vessel area.

For luminal vascular labelling, mice were euthanized with 2.5% (v/v) Avertin and transcardially perfused via peristaltic pump at 2 ml min−1 with the following ice-cold solutions: 8 ml PBS, 10 ml of 5 μg ml−1 of StcE(E447D)–AF647 or SNA–Cy3 (Vector Labs, CL-1303-1), and 8 ml of 4% PFA. Mice were euthanized with 2.5% of their body weight. Avertin and manually perfused with PBS unless noted otherwise. Tissues were extracted and fixed in 4% PFA at 4 °C overnight before preservation in 30% sucrose in PBS. Tissues were sectioned into 40 μm slices using a microtome (Leica). Following a block of slices by blocking 3% normal donkey Serum with 3.0%0.1% Triton X-100 in TBS-T for 1.5 h at room temperature, the slices were put into 4C chambers for overnight use with the following key antibodies: goat anti-CD31. The following day, slices were washed three times with TBS-T, stained with the appropriate Alexa Fluor-conjugated secondary antibodies (1:250, Thermo Fisher Scientific) or Alexa Fluor-conjugated streptavidin (1:1,000, Thermo Fisher Scientific) for 2 h at room temperature, washed three times again, mounted, and coverslipped with Vectashield Hardset Antifade Mounting Medium with DAPI (Vector Labs, H-1500-10). Images were analysed using ImageJ after the images were looked at on the confocal laser- scanning microscope. The vessel area occupied by the marker of interest was divided by the total vessel area to calculate the luminal vascular coverage. Endothelial MFI calculated using CD31+ mask.

Post mortem fresh-frozen brain tissues were obtained from Stanford/VA Aging Clinical Research Center with approval from the Stanford Institutional Review Board and patient consent. The samples used in the study were kept at 80 C until the time of processing, because autopsies were preformed no more than 12 h after death. Group characteristics are summarized in Supplementary Data 2. Individuals in the Alzheimer’s disease group were both clinically diagnosed and pathologically determined to exhibit Alzheimer’s disease brain hallmarks including β-amyloid and tau pathophysiology.

The mice were injected retro-orbitalally with a little bit of HRP type II in PBS. After 30 min, brains had been fixed in a 0.1 Msodium cacodylate buffer with 4% PFA and 5% glutaraldehyde. After washing them with 0.1 M sodium cacodylate, they were cut using a matrix into 1mm-thick sections. Thetical punches were cut and placed in a container with a small amount of hydrogen peroxide in the chamber for 45 min at room temperature. Tissues were washed with TBS overnight, post-fixed in 2% osmium tetroxide and 2.5% potassium ferrocyanide in 0.1 M sodium cacodylate, and en bloc stained with 1% uranyl acetate and Walton’s lead aspartate stain. In the process of dehydration, samples were encapsulated in a substance known as “Epsy resin”. Eighty-nanometre sections were cut using a Leica UC7 ultramicrotome (Leica Microsystems) and collected on formvar-coated 100-mesh copper grids. The grids were post-stained with 3.5% uranyl acetate followed by Sato’s lead citrate. Sections were imaged using a Tecnai 12 120 kV TEM (FEI), and data were recorded using a Rio16 CMOS camera with GWS software (Gatan). The assessment of tight junctions in the images was performed in a blinded manner.