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Spatial profiling of chromatin accessibility in mouse and human tissues


Juvenile mouse mind and pattern preparation

Experimental procedures on juvenile (P21) mice had been performed in accordance with the European directive 2010/63/EU, native Swedish directive L150/SJVFS/2019:9, Saknr L150 and the Karolinska Institutet complementary pointers for procurement and use of laboratory animals, Dnr 1937/03-640. The procedures described right here had been accredited by Stockholms Norra Djurförsöksetiska nämnd, the native committee for moral experiments on laboratory animals in Sweden, 1995/2019 and 7029/2020.

The mouse line Sox10:cre-RCE:LoxP (eGFP), on a C57BL/6xCD1 combined genetic background, was used for experiments on P21 mice. It was generated by crossing Sox10:cre animals51 (Jackson Laboratory, 025807) on a C57BL/6j genetic background with RCE:loxP (enhanced inexperienced fluorescent protein (eGFP)) animals52 (Jackson Laboratory, 32037-JAX) on a C57BL/6xCD1 combined genetic background. Breeding of feminine mice with a hemizygous cre allele with male mice missing the cre allele (whereas the reporter allele was stored in hemizygosity or homozygosity in each feminine and male mice) resulted in labelling of the oligodendrocyte lineage with eGFP. Mice, freed from frequent viral pathogens, ectoparasites, endoparasites and mouse bacterial pathogens, had been housed to a most of 5 mice per cage in individually ventilated cages (IVC Sealsafe GM500, Tecniplast). The cages had been outfitted with hardwood bedding (TAPVEI), nesting materials, shredded paper, gnawing sticks and a cardboard field shelter (Scanbur). Mice obtained common chew weight-reduction plan and water utilizing a water bottle that was modified weekly. Cages had been modified each different week in a laminar air-flow cupboard. Normal housing parameters, resembling relative humidity, temperature and air flow, had been used in accordance with the European conference for the safety of vertebrate animals used for experimental and different scientific functions treaty ETS 123. Particularly, constant relative air humidity and temperature had been set to 50% and 22 °C, and the air high quality was managed with using stand-alone air dealing with models supplemented with a HEPA filter. Husbandry parameters had been monitored utilizing the ScanClime (Scanbur) models. The next mild–darkish cycle was used: daybreak, 6:00–7:00; daylight, 7:00–18:00; nightfall, 18:00–19:00; night time, 19:00–6:00.

Submit-mortem human mind and pattern preparation

One human hippocampus pattern was obtained from the Mind Assortment of the New York State Psychiatric Institute (NYSPI) and Columbia College53. This mind pattern was a contemporary frozen unfixed specimen. All procedures of mind assortment and post-mortem had been performed with Institutional Assessment Board approval and knowledgeable consent from the following of kin. The participant chosen was freed from neuropsychiatric sickness on the idea of our validated psychological post-mortem interview of the following of kin54, died of sudden dying (industrial accident) with quick agonal state (that may have an effect on mind oxygenation if extended), had quick autopsy interval (6.5 h), clear neuropathological examination, unfavorable mind toxicology for psychoactive medication, remedy and alcohol, and good RNA high quality (RNA integrity quantity 8.50).

The anterior hippocampus was dissected from a 2-cm-thick coronal block of the correct hemisphere, and sectioned at a thickness of 10 μm utilizing a cryostat (Leica 3050S). Every of the serial sections of the dentate gyrus area (round 1 cm × 1 cm) had been positioned onto an ultraclean glass slide (Electron Microscopy Sciences, 63478-AS). We took one slide for sequencing and carried out H&E and Nissl staining on two adjoining slides. The entire samples had been saved at −80 °C earlier than use.

Fabrication and meeting of the microfluidic machine

The moulds for microfluidic units had been fabricated within the cleanroom utilizing normal photolithography. We adopted the producer’s pointers to spin-coat SU-8-negative photoresist (SU-2010, SU-2025, Microchem) onto a silicon wafer (C04004, WaferPro). The characteristic heights of the 50-µm-wide and 20-µm-wide microfluidic channel machine had been about 50 µm and 23 µm, respectively. Throughout publicity to ultraviolet mild, chrome photomasks (Entrance Vary Photomasks) had been used. Comfortable lithography was used for the fabrication of polydimethylsiloxane (PDMS) microfluidic units. We combined base and curing agent at a ten:1 ratio and added it over the SU-8 masters. The PDMS was cured (at 65 °C for two h) after degassing in a vacuum (30 min). After solidification, the PDMS slab was minimize out. The outlet and inlet holes had been punched for additional use. We’ve printed a protocol by way of machine fabrication and operation55.

Preparation of tissue slides

Mouse C57 Embryo Sagittal Frozen Sections (MF-104-11-C57) and Human Tonsil Frozen Sections (HF-707) had been bought from Zyagen. Tissues had been snap-frozen in optimum reducing temperature compounds, sectioned (thickness of seven–10 µm) and put on the centre of poly-l-lysine-covered glass slides (63478-AS, Electron Microscopy Sciences).

H&E staining

The frozen slide was warmed at room temperature for 10 min and stuck with 1 ml 4% formaldehyde (10 min). After being washed as soon as with 1× DPBS, the slide was rapidly dipped in water and dried with air. Isopropanol (500 μl) was then added to the slide and incubated for 1 min earlier than being eliminated. After utterly dry within the air, the tissue part was stained with 1 ml haematoxylin (Sigma-Aldrich) for 7 min and cleaned in deionized water. The slide was then incubated in 1 ml bluing reagent (0.3% acid alcohol, Sigma-Aldrich) for two min and rinsed in deionized water. Lastly, the tissue slide was stained with 1 ml eosin (Sigma-Aldrich) for two min and cleaned in deionized water.

Preparation of the transposome

Unloaded Tn5 transposase (C01070010) was bought from Diagenode, and the transposome was assembled in accordance with the producer’s pointers. The oligos used for transposome meeting had been as follows: Tn5MErev, 5′-/5Phos/CTGTCTCTTATACACATCT-3′; Tn5ME-A, 5′-/5Phos/CATCGGCGTACGACTAGATGTGTATAAGAGACAG-3′; Tn5ME-B, 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′.

DNA oligos, DNA barcode sequences and different key reagents

Lists of the DNA oligos that had been used for sequencing library development and PCR (Supplementary Desk 3), DNA barcode sequences (Supplementary Desk 3) and all different key reagents (Supplementary Desk 4) are supplied.

Spatial-ATAC-seq profiling

As we proceeded to develop spatial-ATAC-seq, we went by a number of variations of chemistry to optimize the protocol to realize a excessive yield and a excessive signal-to-noise ratio for the mapping of tissue sections (Supplementary Fig. 2a). First, a set of fifty DNA oligomers containing each barcode A and adapter had been launched in microchannels to a tissue part for in situ transposition, however the effectivity was low due partly to restricted quantities of Tn5 DNA within the microchannels. To deal with this concern, we performed bulk transposition adopted by two ligation steps to introduce spatial barcodes A and B. We additionally optimized the fixation situation by decreasing the formaldehyde focus from 4% in chemistry V1 to 0.2%. Moreover, we examined the sensitivity of various Tn5 transposase enzymes (Diagenode (C01070010) versus Lucigen (TNP92110)).

Within the optimized spatial-ATAC-seq protocol, the frozen slide was warmed at room temperature for 10 min. The tissue was then fastened with formaldehyde (0.2% for five min) and quenched with glycine (1.25 M for five min) at room temperature. After fixation, the tissue was washed twice with 1 ml 1× DPBS and cleaned in deionized water. The tissue part was then permeabilized with 500 µl lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.01% Tween-20, 0.01% NP-40, 0.001% digitonin, 1% BSA) for 15 min and was washed with 500 µl wash buffer(10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1% BSA, 0.1% Tween-20) for five min. Then, 100 µl transposition combine (50 µl 2× tagmentation buffer, 33 µl 1× DPBS, 1 µl 10% Tween-20, 1 µl 1% digitonin, 5 µl transposome, 10 µl nuclease-free H2O) was added adopted by incubation at 37 °C for 30 min. After eradicating the transposition combine, 500 µl 40 mM EDTA was added for incubation at room temperature for five min to cease transposition. Lastly, the EDTA was eliminated, and the tissue part was washed with 500 µl 1× NEBuffer 3.1 for five min.

For barcode A in situ ligation, the primary PDMS slab was used to cowl the area of curiosity, the bright-field picture was taken utilizing a ×10 goal (Thermo Fisher Scientific, EVOS FL Auto microscope (AMAFD1000), EVOS FL Auto Software program (REV 32044)) for additional alignment. The tissue slide and PDMS machine had been then clamped with an acrylic clamp. First, DNA barcode A was annealed with ligation linker 1: 10 μl of every DNA barcode A (100 μM), 10 μl of ligation linker (100 μM) and 20 μl of two× annealing buffer (20 mM Tris, pH 7.5–8.0, 100 mM NaCl, 2 mM EDTA) had been added collectively and combined properly. Then, 5 μl ligation response resolution (50 tubes) was ready by including 2 μl of ligation combine (72.4 μl of RNase-free water, 27 μl of T4 DNA ligase buffer, 11 μl T4 DNA ligase, 5.4 μl of 5% Triton X-100), 2 μl of 1× NEBuffer 3.1 and 1 μl of every annealed DNA barcode A (A1–A50, 25 μM) and loaded into every of the 50 channels below a vacuum. The chip was stored in a moist field for incubation (37 °C, 30 min). After flowing by 1× NEBuffer 3.1 for laundry (5 min), the clamp and PDMS had been eliminated. The slide was rapidly dipped in water and dried with air.

For barcode B in situ ligation, the second PDMS slab with channels perpendicular to the primary PDMS was connected to the dried slide fastidiously. A bright-field picture was taken and the acrylic clamp was used to press the PDMS in opposition to the tissue. The annealing of DNA barcode B with ligation linker 2 was carried out the identical as described above for the annealing of DNA barcode A and ligation linker 1. The preparation and addition of the ligation response resolution for DNA barcode B (B1–B50, 25 μM) had been additionally the identical as described for DNA barcode A (A1–A50, 25 μM). The chip was stored in a moist field for incubation (37 °C for 30 min). After flowing by 1× DPBS for laundry (5 min), the clamp and PDMS had been eliminated, the tissue part was dipped in water and dried with air. The ultimate bright-field picture of the tissue was taken.

For tissue digestion, the area of curiosity of the tissue was lined with a sq. PDMS properly gasket, and 100 μl reverse cross-linking resolution (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1% SDS, 200 mM NaCl, 0.4 mg ml−1 proteinase Ok) was loaded into it. The lysis was performed in a moist field (58 °C, 2 h). The ultimate tissue lysate was collected right into a 200 μl PCR tube for incubation with rotation (65 °C, in a single day).

For library development, the lysate was first purified utilizing the Zymo DNA Clear & Concentrator-5 package and eluted into 20 μl of DNA elution buffer, adopted by mixing with the PCR resolution (2.5 µl 25 µM new P5 PCR primer, 2.5 µl 25 µM Ad2 primer, 25 µl 2× NEBNext Grasp Combine). Then, PCR was carried out utilizing the next program: 72 °C for five min; 98 °C for 30 s; after which cycled 5 instances at 98 °C for 10 s, 63 °C for 10 s and 72 °C for 1 min. To find out further cycles, 5 µl of the pre-amplified combination was first combined with the qPCR resolution (0.5 µl 25 µM new P5 PCR primer, 0.5 µl 25 µM Ad2 primer, 0.24 µl 25× SYBR Inexperienced, 5 µl 2× NEBNext Grasp Combine, 3.76 µl nuclease-free H2O). Then, the qPCR response was carried out below the next situations: 98 °C for 30 s; after which 20 cycles of 98 °C for 10 s, 63 °C for 10 s and 72 °C for 1 min. Lastly, the remaining 45 µl of the pre-amplified DNA was amplified by working the required variety of further cycles of PCR (the cycles wanted to achieve 1/3 of the saturated sign in qPCR).

To take away PCR primer residues, the ultimate PCR product was purified utilizing 1× Ampure XP beads (45 µl) in accordance with the usual protocol and eluted into 20 µl nuclease-free H2O. Earlier than sequencing, an Agilent Bioanalyzer Excessive Sensitivity Chip was used to quantify the focus and measurement distribution of the library. Subsequent-generation sequencing was carried out utilizing the Illumina HiSeq 4000 or NovaSeq 6000 sequencer (paired-end 150 bp mode with customized learn 1 primer).

Information preprocessing

Two fixed linker sequences (linker 1 and linker 2) had been used to filter learn 1, and the filtered sequences had been reworked to Cell Ranger ATAC format (10x Genomics). The genome sequences had been included within the new learn 1, barcodes A and barcodes B had been included within the new learn 2. The ensuing fastq recordsdata had been aligned to the mouse reference (mm10) or human reference (GRCh38) genome, filtered to take away duplicates and counted utilizing Cell Ranger ATAC v.1.2. The BED-like fragments recordsdata had been generated for downstream evaluation. The fragments file incorporates fragments of data on the genome and tissue location (barcode A × barcode B). A preprocessing pipeline we developed utilizing Snakemake workflow administration system (v5.28.0) is out there at GitHub (https://github.com/dyxmvp/Spatial_ATAC-seq).

Information visualization

We first recognized pixels on tissue samples by guide choice from microscopy photographs utilizing Adobe Illustrator (v.25.4.3) (https://github.com/rongfan8/DBiT-seq), and a customized Python script was used to generate metadata recordsdata that had been suitable with the Seurat workflow for spatial datasets.

The fragment file was learn into ArchR as a tile matrix with a genome binning measurement of 5 kb, and pixels that weren’t on the tissue had been eliminated on the idea of the metadata file generated within the earlier step. Information normalization and dimensionality discount was performed utilizing iterative latent semantic indexing (iterations = 2, decision = 0.2, varFeatures = 25000, dimsToUse = 1:30, n.begin = 10), adopted by graph clustering and UMAP embedding (nNeighbors = 30, metric = cosine, minDist = 0.5)14.

The Gene Rating mannequin in ArchR was used to generate the gene accessibility rating. A gene rating matrix was generated for downstream evaluation. The getMarkerFeatures and getMarkers perform in ArchR (testMethod = “wilcoxon”, cutOff = “FDR < = 0.05”) was used to determine the marker areas/genes for every cluster, and the marker genes had been mentioned within the manuscript as a result of they had been recognized as one of many prime differential genes between clusters, and so they had been additionally recognized within the literature. Gene-score imputation was applied with addImputeWeights for information visualization. The enrichGO perform within the clusterProfiler package deal was used for GO enrichment evaluation (qvalueCutoff = 0.05)56. For spatial information visualization, outcomes obtained in ArchR had been loaded into Seurat v.3.2.3 to map the info again to the tissue part57,58. For higher visualization, we scaled the dimensions of the pixels utilizing the ‘pt.measurement.issue’ parameter within the Seurat package deal (Prolonged Information Fig. 8c).

Genome browser tracks had been plotted utilizing the plotBrowserTrack perform in ArchR. Spatial-ATAC-seq information had been normalized to the really useful and default worth (normMethod = “ReadsInTSS”), which concurrently normalizes tracks primarily based on sequencing depth and pattern information high quality. Blue-coloured genes are on the minus strand and red-coloured genes are on the plus strand. The loops are the hyperlinks between a peak and a gene, and the color exhibits the Pearson correlation between peak accessibility and gene expression. Peaks had been known as with macs2 utilizing addReproduciblePeakSet perform in ArchR.

To mission bulk ATAC-seq information, we downloaded uncooked sequencing information aligned to the mm10 genome (BAM recordsdata) from ENCODE. After counting the reads in 5 kb tiled genomes utilizing the getCounts perform in chromVAR59, the ENCODE ATAC-seq information had been subsampled in pseudo single cells (n = 250) and had been projected onto spatial-ATAC UMAPs utilizing the projectBulkATAC perform in ArchR.

Cell sort identification and pseudo-scRNA-seq profiles had been added by integration with scRNA-seq reference information20. The FindTransferAnchors perform (Seurat v.3.2 package deal) was used to align pixels from spatial-ATAC-seq with cells from scRNA-seq by evaluating the spatial-ATAC-seq gene rating matrix with the scRNA-seq gene expression matrix. The GeneIntegrationMatrix perform in ArchR was used so as to add cell identities and pseudo-scRNA-seq profiles.

Pseudobulk group coverages primarily based on cluster identities had been generated utilizing the addGroupCoverages perform and used for peak calling with macs2 utilizing the addReproduciblePeakSet perform in ArchR. To compute per-cell motif exercise, chromVAR59 was run with addDeviationsMatrix utilizing the cisbp motif set after a background peak set was generated utilizing addBgdPeaks. Cell-type-specific marker peaks had been recognized utilizing the getMarkerFeatures (bias = c(“TSSEnrichment”, “log10(nFrags)”, testMethod = “wilcoxon”) and getMarkers (cutOff = “FDR < = 0.05 & Log2FC > = 0.1”) capabilities.

Pseudotemporal reconstruction was applied by trajectory evaluation utilizing ArchR. We used the cell-type definitions from label switch as described above. A trajectory spine was first created within the type of an ordered vector of cell group labels. We then used the addTrajectory perform to create a trajectory, and added the pseudotime to Seurat spatial object to map the info again to the tissue part. Dynamics for chosen gene rating alongside the pseudotime had been plotted with the plotTrajectory perform utilizing the default values.

Correlation evaluation was performed by calculating the Pearson correlation coefficient in R. P values had been calculated utilizing the cor.check perform and had been adjusted for a number of comparisons utilizing the Benjamini–Hochberg technique60.

Reporting abstract

Additional data on analysis design is out there within the Nature Analysis Reporting Abstract linked to this text.

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