Preparation of tissue slides
Mouse C57 embryo sagittal frozen sections (no. MF-104-13-C57) were purchased from Zyagen. Freshly harvested E13 mouse embryos were snap-frozen in optimal cutting temperature compounds and sectioned at 7−10 μm thickness. Tissue sections were collected on poly-l-lysine-coated glass slides (Electron Microscopy Sciences, no. 63478-AS).
Juvenile mouse brain tissue (P21−P22) was obtained from the Sox10:Cre-RCE:LoxP enhanced green fluorescent protein (eGFP) line on a C57BL/6xCD1 mixed genetic background maintained at Karolinska Institutet. The line was first generated by crossing Sox10:Cre animals (The Jackson Laboratory, no. 025807) with RCE:loxP (eGFP) animals (The Jackson Laboratory, no. 032037-JAX). This was established and maintained by breeding males lacking the Cre allele with females carrying a hemizygous Cre allele; the reporter allele was maintained in homozygosity or hemizygosity in both males and females. This resulted in specific labelling of oligodendrocyte lineage with eGFP. All animals were free from mouse bacterial and viral pathogens, ectoparasites and endoparasites. The following light/dark cycle was maintained for the mice: dawn, 06:00–07:00; daylight, 07:00–18:00; dusk, 18:00–19:00; night, 19:00–06:00. Mice were housed in individually ventilated cages at a maximum number of five per cage (IVC sealsafe GM500, tecniplast). General housing parameters including temperature, ventilation and relative humidity followed the European Convention for the Protection of Vertebrate Animals used for experimental and other scientific purposes. Air quality was controlled using stand-alone air-handling units equipped with a high-efficiency particulate air filter. Relative air humidity was consistently 55 ± 10%, at a temperature of 22 °C. Husbandry parameters were monitored with ScanClime (Scanbur) units. The cages contained a card box shelter, gnawing sticks and nesting material (Scanbur), placed on hardwood bedding (TAPVEI). Mice were provided with a regular chow diet, and water was supplied by water bottle and changed weekly. Cages were changed every 2 weeks in a laminar air-flow cabinet.
Mice were sacrificed at P21/P22 (both sexes were used) by anaesthesia with ketamine (120 mg kg–1 body weight) and xylazine (14 mg kg–1 body weight), followed by transcranial perfusion with cold oxygenated artificial cerebrospinal fluid (87 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 75 mM sucrose, 20 mM glucose, 1 mM CaCl2*2H2O and 2 mM MgSO4*7H2O in distilled H2O). Following isolation, brains were maintained for a minimal time period in artificial cerebrospinal fluid until embedding in Tissue-Tek O.C.T. compound (Sakura) and snap-freezing using a mixture of dry ice and ethanol. Coronal cryosections of 10 μm were mounted on poly-l-lysine-coated glass slides (no. 63478-AS, Electron Microscopy Sciences) or 2 × 3 square inch glass slides (AtlasXomics).
All experimental procedures were conducted following European directive no. 2010/63/EU, local Swedish directive no. L150/SJVFS/2019:9, Saknr no. L150 and Karolinska Institutet complementary guidelines for procurement and use of laboratory animals, no. Dnr. 1937/03-640. All procedures described were approved by the local committee for ethical experiments on laboratory animals in Sweden (Stockholms Norra Djurförsöksetiska nämnd, nos. 1995/2019 and 7029/2020).
Human brain tissue was obtained from the brain collection of the New York State Psychiatric Institute at Columbia University, which includes brain samples from the Republic of Macedonia. Brain tissue collection was conducted with New York State Psychiatric Institute Institutional Review Board approval, and informed consent obtained from next of kin who agreed to donate the brains and participate in psychological autopsy interviews.
We analysed brain hippocampus tissue from a 31-year-old Caucasian male individual, with no psychiatric or neurological diagnosis, who had died of a traumatic accident and had a high level of global functioning before death as measured by global assessment scale51 score, which was 90 (scoring 1–100, with 100 the highest function), and with toxicology negative for psychotropic medications and drugs. Postmortem interval (time from demise to brain collection) was 6.5 h.
The anterior hippocampal region was dissected from a fresh-frozen coronal section (20-mm thickness) of the right brain hemisphere. The dentate gyrus region (around 10 × 10 mm2) of the anterior hippocampal region was selected. Cryosections of 10 μm were collected on poly-l-lysine-coated glass slides (no. 63478-AS, Electron Microscopy Sciences). Samples were stored at −80 °C until further use.
Preparation of transposome
Unloaded Tn5 transposase (no. C01070010) and pA-Tn5 (no. C01070002) were purchased from Diagenode, and the transposome was assembled following the manufacturer’s guidelines. The oligos applied for transposome assembly were:
Tn5MErev, 5′-/5Phos/CTGTCTCTTATACACATCT-3′ and
DNA barcode sequences, DNA oligos and other key reagents
DNA oligos used for PCR and library construction are shown in Supplementary Table 4. All DNA barcode sequences are provided in Supplementary Tables 5 and 6 and all other chemicals and reagents in Supplementary Table 7.
Fabrication of PDMS microfluidic device
Chrome photomasks were purchased from Front Range Photomasks, with a channel width of either 20 or 50 μm. The moulds for polydimethylsiloxane (PDMS) microfluidic devices were fabricated using standard photolithography. The manufacturer’s guidelines were followed to spin-coat SU-8-negative photoresist (nos. SU-2025 and SU-2010, Microchem) onto a silicon wafer (no. C04004, WaferPro). The heights of the features were about 20 and 50 μm for 20- and 50-μm-wide devices, respectively. PDMS microfluidic devices were fabricated using the SU-8 moulds. We mixed the curing and base agents in a 1:10 ratio and poured the mixture into the moulds. After degassing for 30 min the mixture was cured at 70 °C for 2 h. Solidified PDMS was extracted for further use. We have published a detailed protocol for the fabrication and preparation of the PDMS device52.
Frozen tissue slides were thawed for 10 min at room temperature. Tissue was fixed with formaldehyde (0.2%, with 0.05 U μl–1 RNase Inhibitor) for 5 min and quenched with 1.25 M glycine for a further 5 min. After fixation, tissue was washed twice with 1 ml of 0.5× DPBS-RI and cleaned with deionized (DI) H2O.
Tissue permeabilization was carried out with 200 μl of lysis buffer (3 mM MgCl2, 0.01% Tween-20, 10 mM Tris-HCl pH 7.4, 0.01% NP40, 10 mM NaCl, 1% bovine serum albumin (BSA), 0.001% digitonin, 0.05 U μl–1 RNase inhibitor) for 15 min and washed twice with 200 μl of wash buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1% BSA, 0.1% Tween-20) for 5 min. Transposition mix (5 μl of home-made transposome, 33 μl of 1× DPBS, 50 μl of 2× Tagmentation buffer, 1 μl of 1% digitonin, 1 μl of 10% Tween-20, 0.05 U μl–1 RNase Inhibitor, 10 μl of nuclease-free H2O) was added with incubation at 37 °C for 30 min. Next, 200 μl of 40 mM EDTA with 0.05 U μl–1 RNase inhibitor was added with incubation for 5 min at room temperature, to stop transposition. Finally, tissue sections were washed twice with 200 μl of 0.5× PBS-RI for 5 min and cleaned with DI water.
For RT, the following mixture was used: 12.5 μl of 5× RT buffer, 4.5 μl of RNase-free water, 0.4 μl of RNase inhibitor, 0.8 μl of Superase In RNase inhibitor, 3.1 μl of 10 mM deoxynucleotide triphosphate each, 6.2 μl of Maxima H Minus Reverse Transcriptase, 25 μl of 0.5× PBS-RI and 10 μl of RT primer. Tissues were incubated for 30 min at room temperature, then at 42°C for 90 min in a wet box. After the RT reaction, tissues were washed with 1× NEBuffer 3.1 and 1% RNase inhibitor for 5 min.
For first barcode (barcode A) in situ ligation, the first PDMS chip was covered to the tissue region of interest. For alignment purposes, a 10× objective (Thermo Fisher Scientific, EVOS FL Auto microscope no. AMF7000, EVOS FL Auto 2 Software Revision 2.0.2094.0) was used to take a brightfield image. The PDMS device and tissue slide were clamped tightly with a home-made acrylic clamp. Barcode A was first annealed with ligation linker 1, 10 μl of 100 μM ligation linker, 10 μl of 100 μM each barcode A and 20 μl of 2× annealing buffer (20 mM Tris pH 7.5–8.0, 100 mM NaCl, 2 mM EDTA) and mixed well. For each channel, 5 μl of ligation master mixture was prepared with 2 μl of ligation mix (27 μl of T4 DNA ligase buffer, 72.4 μl of RNase-free water, 5.4 μl of 5% Triton X-100, 11 μl of T4 DNA ligase), 2 μl of 1× NEBuffer 3.1 and 1 μl of each annealed DNA barcode A (A1−A50/A100, 25 μM). Vacuum was used to load the ligation master mixture into 50 channels of the device, followed by incubation at 37 °C for 30 min in a wet box. The PDMS chip and clamp were removed after washing with 1× NEBuffer 3.1 for 5 min. The slide was washed with water and dried in air.
For second barcode (barcode B) in situ ligation, the second PDMS chip was covered to the slide and a further brightfield image taken with the 10× objective. An acrylic clamp was applied to clamp the PDMS and tissue slide together. Annealing of barcodes B (B1−B50/B100, 25 μM) and preparation of the ligation master mix were carried out as for barcodes A. The device was incubated at 37 °C for 30 min in a wet box. The PDMS chip and clamp were removed after washing with 1× DPBS with SUPERase In RNase inhibitor for 5 min. The slide was washed with water and dried in air. A brightfield image was then taken for further alignment.
For tissue lysis, the region of interest was digested with 100 μl of reverse crosslinking mixture (0.4 mg ml–1 proteinase K, 1 mM EDTA, 50 mM Tris-HCl pH 8.0, 200 mM NaCl, 1% SDS) at 58 °C for 2 h in a wet box. The lysate was collected in a 1.5 m tube and incubated at 65 °C overnight.
For DNA and cDNA separation, the lysate was purified with Zymo DNA Clean & Concentrator-5 and eluted to 100 μl of RNase-free water. The 1× B&W buffer with 0.05% Tween-20 was used to wash 40 μl of Dynabeads MyOne Streptavidin C1 beads three times. Then, 100 μl of 2× B&W buffer with 2.5 μl of SUPERase In RNase inhibitor was used to resuspend the beads, which were then mixed with the lysate and allowed to bind at room temperature for 1 h with agitation. A magnet was used to separate beads and supernatant in the lysate.
The supernatant was removed for ATAC library construction, then purified with Zymo DNA Clean & Concentrator-5 and eluted to 20 μl of RNase-free water. PCR solution (25 μl of 2× NEBNext Master Mix, 2.5 μl of 25 μM indexed i7 primer, 2.5 μl of 25 μM P5 PCR primer) was added and mixed well. PCR was first performed with the following programme: 72 °C for 5 min, 98 °C for 30 s and cycling at 98 °C for 10 s, 63 °C for 30 s and 72 °C for 1 min, five times. To determine additional cycles, the pre-amplified mixture (5 μl) was mixed with quantitative PCR (qPCR) solution (5 μl of 2× NEBNext Master Mix, 0.24 μl of 25× SYBR Green, 0.5 μl of 25 μM new P5 PCR primer, 3.76 μl of nuclease-free H2O, 0.5 μl of 25 μM indexed i7 primer). The qPCR reaction was then carried out with the following programme: 98 °C for 30 s with cycling at 98 °C for 10 s, 63 °C for 30 s and 72 °C for 1 min, 20 times. The remaining pre-amplified DNA (45 μl) was amplified by running additional cycles as determined by qPCR (to reach one-third of saturated signal). The final PCR product was purified by 1× Ampure XP beads (45 μl) and eluted in 20 μl of nuclease-free H2O.
The beads were used for cDNA library construction. They were first washed twice with 400 μl of 1× B&W buffer with 0.05% Tween-20 and once with 10 mM Tris pH 8.0 containing 0.1% Tween-20. Streptavidin beads with bound cDNA molecules were resuspended in a TSO solution (22 μl of 10 mM deoxynucleotide triphosphate each, 44 μl of 5× Maxima RT buffer, 44 μl of 20% Ficoll PM-400 solution, 88 μl of RNase-free water, 5.5 μl of 100 uM template switch primer (AAGCAGTGGTATCAACGCAGAGTGAATrGrG+G), 11 μl of Maxima H Minus Reverse Transcriptase, 5.5 μl of RNase Inhibitor). The beads were incubated at room temperature for 30 min and then at 42 °C for 90 min, with gentle shaking. After washing beads once with 400 μl of 10 mM Tris and 0.1% Tween-20 and once with water, they were resuspended in a PCR solution (110 μl of 2× Kapa HiFi HotStart Master Mix, 8.8 μl of 10 μM primers 1 and 2, 92.4 μl of RNase-free water). PCR thermocycling was carried out using the following programme: 95 °C for 3 min and cycling at 98 °C for 20 s, 65 °C for 45 s and 72 °C for 3 min, five times. After five cycles, beads were removed from the PCR solution and 25× SYBR Green was added at 1× concentration. Samples were again placed in a qPCR machine with the following thermocycling conditions: 95 °C for 3 min, cycling at 98 °C for 20 s, 65 °C for 20 s and 72 °C for 3 min, 15 times, followed by 5 min at 72 °C. The reaction was removed once the qPCR signal began to plateau. The PCR product was purified with 0.8× Ampure XP beads and eluted in 20 μl of nuclease-free H2O.
A Nextera XT Library Prep Kit was used for library preparation. Purified cDNA (1 ng) was diluted in RNase-free water to a total volume of 5 μl, then 10 μl of Tagment DNA buffer and 5 μl of Amplicon Tagment mix were added with incubation at 55 °C for 5 min; 5 μl of NT buffer was then added, with incubation at room temperature for 5 min. PCR master solution (15 μl of PCR master mix, 1 μl of 10 μM P5 primer, 1 μl of 10 μM indexed P7 primer, 8 μl of RNase-free water) was added and the PCR reaction performed with the following programme: 95 °C for 30 s, cycling at 95 °C for 10 s, 55 °C for 30 s, 72 °C for 30 s and 72 °C for 5 min, for 12 cycles. The PCR product was purified with 0.7× Ampure XP beads to obtain the library.
An Agilent Bioanalyzer High Sensitivity Chip was used to determine size distribution and concentration of the library before sequencing. NGS was conducted on an Illumina NovaSeq 6000 sequencer (paired-end, 150-base-pair mode).
The frozen tissue slide was thawed for 10 min at room temperature. Tissue was fixed with formaldehyde (0.2%, with 0.05 U μl–1 RNase inhibitor) for 5 min and quenched with 1.25 M glycine for a further 5 min. After fixation, the tissue was washed twice with 1 ml of wash buffer (150 mM NaCl, 20 mM HEPES pH 7.5, one tablet of protease inhibitor cocktail, 0.5 mM Spermidine) and dipped in DI water. The tissue section was permeabilized with NP40-digitonin wash buffer (0.01% digitonin, 0.01% NP40 in wash buffer) for 5 min. The primary antibody (1:50 dilution with antibody buffer (0.001% BSA, 2 mM EDTA in NP40-digitonin wash buffer) was added with incubation at 4 °C overnight. The secondary antibody (guinea pig anti-rabbit IgG, 1:50 dilution with NP40-digitonin wash buffer) was added with incubation for 30 min at room temperature. The tissue was then washed with wash buffer for 5 min. A 1:100 dilution of pA-Tn5 adaptor complex in 300-wash buffer (one tablet of Protease inhibitor cocktail, 300 mM NaCl, 0.5 mM Spermidine, 20 mM HEPES pH 7.5) was added with incubation at room temperature for 1 h, followed by a 5 min wash with 300-wash buffer. Tagmentation buffer (10 mM MgCl2 in 300-wash buffer) was added with incuation at 37 °C for 1 h. Next, 40 mM EDTA with 0.05 U μl–1 RNase inhibitor was added with incubation at room temperature for 5 min to stop tagmentation. Tissue was washed twice with 0.5× DPBS-RI for 5 min for further use.
For RT, two ligations and bead separation the protocols were as for spatial ATAC–RNA-seq. For construction of the CUT&Tag library, the supernatant was purified with Zymo DNA Clean & Concentrator-5 and eluted to 20 μl of RNase-free water. PCR solution (2 μl each of 10 μM P5 PCR primer and indexed i7 primer, 25 μl of NEBNext Master Mix) was added and mixed well. PCR was performed with the following programme: 58 °C for 5 min, incubation at 72 °C for 5 min and 98 °C for 30 s then cycling at 98 °C for 10 s, with incubation at 60 °C for 10 s 12 times and a final incubation at 72 °C for 1 min. The PCR product was purified by 1.3× Ampure XP beads using the standard protocol and eluted in 20 μl of nuclease-free H2O. cDNA library construction followed the spatial ATAC–RNA-seq protocol.
An Agilent Bioanalyzer High Sensitivity Chip was used to determine size distribution and concentration of the library before sequencing. NGS was conducted on an Illumina NovaSeq 6000 sequencer (paired-end, 150-base-pair mode).
For ATAC and CUT&Tag data, linkers 1 and 2 were used to filter read 2 and sequences were converted to Cell Ranger ATAC v.1.2 format (10X Genomics). Genome sequences were in the newly formed read 1, and barcodes A and B were included in newly formed read 2. Human reference (GRCh38) or mouse reference (GRCm38) was used to align fastq files. The BED-like fragments thus obtained were used to conduct downstream analysis. The fragments file includes fragments of information on spatial locations (barcode A × barcode B) and the genome.
For RNA data, read 2 was refined to extract barcode A, barcode B and UMI. ST pipeline v.1.7.2 (ref. 53) was used to map the processed read 1 against the mouse genome (GRCm38) or human genome (GRCh38), which created the gene matrix for downstream analysis that contains information on genes and spatial locations (barcode A × barcode B).
Data clustering and visualization
We first identified the location of pixels on tissue from the brightfield image using MATLAB 2020b (https://github.com/edicliuyang/Hiplex_proteome).
Signac v.1.8 (ref. 54) was loaded in R v.4.1. ATAC, CUT&Tag and RNA matrices were read into Signac v.1.8 (ref. 54). The ‘DefaultAssay’ function was used for the RNA assay. For RNA data visualization, the feature was set to 3,000 with the ‘FindVariableFeatures’ function, then data were normalized using the ‘SCTransform’ function. Normalized RNA data were clustered and RNA UMAP was built. The DefaultAssay function was applied to the ATAC/CUT&Tag assay. For ATAC/CUT&Tag data visualization, minimum cutoff was set with the ‘FindTopFeatures’ function. Data were normalized and dimensionally reduced using latent semantic indexing, then ATAC/CUT&Tag data were clustered and ATAC/CUT&Tag UMAP was built.
The DefaultAssay function was used for the joint ATAC/CUT&Tag and RNA assay. For visualization of joint ATAC/CUT&Tag and RNA data55, the ‘FindMultiModalNeighbors’ function was used. The reduction list was set to (‘pca’, ‘lsi’), the dimensions list was set to that for RNA and ATAC/CUT&Tag, the modality weight.name was set to RNA weight and the joint UMAP was built.
To plot the above-generated UMAPs together, DefaultAssay was set to RNA and the UMAPs for ATAC/CUT&Tag, RNA or joint ATAC/CUT&Tag and RNA were visualized separately using ‘DimPlot’.
In regard to RNA spatial data visualization, the gene matrix obtained from RNA was loaded into Seurat v.4.1 (ref. 56) as a Seurat object, and RNA metadata obtained from Signac were read into the Seurat object. All spatial maps were then plotted with the ‘SpatialPlot’ function.
In regard to ATAC/CUT&Tag spatial data visualization, the fragment file obtained from ATAC/CUT&Tag was read into ArchR v.1.0.1 (ref. 13) as an ArchRProject and the ATAC/CUT&Tag metadata obtained from Signac were read into the ArchRProject. The data from ArchRProject were normalized and dimensionally reduced using iterative latent semantic indexing. For GAS and CSS calculation we used the Gene Score model in ArchR. A gene score matrix was obtained for downstream analysis. The ‘getMarkerFeatures’ and ‘getMarkers’ functions in ArchR (testMethod = “Wilcoxon”, cutOff = “FDR <= 0.05”, groupBy = “seurat_cluster”) were used to find the marker genes/regions for each cluster. To visualize spatial data, results obtained from ArchR were input to Seurat v.4.1 to map the data back to the tissue. Pixel size was scaled using the ‘pt.size.factor’ parameter in the Seurat package for better visualization.
For peak-to-gene links we input RNA Seurat object using the ‘addGeneIntegrationMatrix’ function in ArchR, then peak-to-gene links were drawn with the ‘addPeak2GeneLinks’ function. Co-accessibility of peaks was calculated using the ‘addCoAccessibility’ function in ArchR.
Integrative data analysis and cell type identification
Seurat v.4.1 (ref. 56) was used for RNA data integration and cell type identification, and the ‘SCTransform’ function to normalize our spatial RNA and scRNA-seq data. The ‘SelectIntegrationFeatures’ function was used to obtain features common to the two datasets. The ‘FindIntegrationAnchors’ function was applied to find anchors, and the ‘IntegrateData’ function to create an integrated dataset through the identified anchors. The obtained integrated dataset was clustered, showing a good match between our spatial RNA and scRNA-seq data. The ‘FindTransferAnchors’ function was used to find transfer anchors, which were then used to conduct label transfer with the ‘TransferData’ function (if more than one cell type was presented in one pixel, the major cell type was assigned).
Signac v.1.8 and Seurat v.4.1 were used for integration of our ATAC/CUT&Tag and scATAC-seq/scCUT&Tag data. The scATAC-seq/scCUT&Tag data were quantified according to our ATAC/CUT&Tag data to ensure that there were features common across both datasets. The FindIntegrationAnchors function (reduction = “rlsi”) was used to identify anchors between the two datasets. The ‘IntegrateEmbeddings’ function was used to obtain an integrated dataset through the identified anchors. The obtained integrated dataset was clustered, showing a good match between our spatial ATAC/CUT&Tag and scATAC-seq/scCUT&Tag data. For ATAC data, the FindTransferAnchors function was used to find transfer anchors, which were then used to map scATAC-seq to our spatial ATAC data with the ‘MapQuery’ function.
ArchR v.1.0.1 was used for cell type identification for our ATAC/CUT&Tag data from scRNA-seq data. The gene score matrix of our ATAC/CUT&Tag was compared with the gene expression matrix from scRNA-seq, and aligned pixels from our ATAC/CUT&Tag data with cells from scRNA-seq. The function ‘GeneIntegrationMatrix’ was used to add pseudo-scRNA-seq profiles and cell identities.
Correlation of CSS/GAS and gene expression
Correlation analysis was performed for different tissue regions. The mouse brain hemisphere was separated into seven clusters (corpus callosum, striatum, superficial cortical layer, deeper cortical layer, lateral ventricle, lateral septal nucleus and others) according to RNA clusters and anatomical annotation, and named ‘tissue_clusters’. For Fig. 4a–c, the ‘FindMarkers’ function (settings: min.pct = 0.25, logfc.threshold = 0.25) was used to calculate our RNA data, and genes with adjusted P < 10−5 selected as marker genes. The getMarkerFeatures function (settings: groupBy = “tissue_clusters”) was applied to calculate the GAS or CSS of genes from the marker gene list (cutOff = “FDR <= 0.05” & (cutOff = “Log2FC >= 0.1” or cutOff = “Log2FC <= −0.1”)). If avg_log2FC > 0 (RNA) and Log2FC > 0 (CUT&Tag) for a specific gene, it showed in quadrant I. GO enrichment analysis was conducted with the ‘enrichGO’ function (qvalueCutoff = 0.05) in the clusterProfiler v.4.2 package25.
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.