GoKawiilTissue acquisition and processing for joint single-nucleus multiome profiling and spatial transcriptomics
Snap-frozen decidual and basal plate samples were obtained from the existing placenta tissue banks at Stanford University and University of California, San Francisco (Supplementary Tables 1 and 4). All samples were collected with written informed consent. Tissues were derived from women undergoing elective termination of presumed normal pregnancies (first and second-trimester samples; no known or predicted fetal chromosomal abnormalities, Extended Data Fig. 1c) or after term delivery (≥37 gestational weeks). For term samples, clinical records were reviewed to exclude placenta-associated complications (for example, chorioamnionitis); cases with NICU admission and preterm premature rupture of membranes were also excluded. Fresh placental tissues were grossly inspected and dissected under a microscope (Leica Microsystems) by pathologists. Decidua basalis was micro-dissected on ice from the MFI and distinguished from decidua parietalis and capsularis on the basis of characteristic histological and morphological features (Extended Data Fig. 1a,b). Dissected tissues were sequentially washed to remove residual blood cells in DMEM/H-21 medium, supplemented with 12.5% FBS (Hyclone), 1% L-glutamine (Atlanta Biologicals), 1% penicillin/streptomycin and 0.1% gentamicin and cold 1× PBS (Gibco, Thermofisher). Samples used for single-cell or spatial transcriptomic profiling were flash-frozen in liquid nitrogen and stored at −80 °C until processing. RNA quality was assessed from adjacent cryosections using a Bioanalyzer or Tapestation. For fresh frozen samples, only samples with RIN ≥ 7.0 were included.
Isolation of single nucleus from snap-frozen tissues
Single nuclei were isolated from snap-frozen tissues as previously described61 with modification. In brief, tissues were ground on dry ice, and 30–50 mg was homogenized into a pre-chilled 7 ml PYREX dounce homogenizer (Corning Life Science). Tissue was homogenized in 2 ml ice-cold buffer (250 mM sucrose, 0.3% NP-40, 5 mM MgCl 2 , 25 mM KCl, 10 mM Tris-HCl pH 7.8) supplemented with protease inhibitors (Roche, cOmplete) and 0.6 U µl−1 Ribolock RNase inhibitor (thermofisher). Debris was removed by 40 μm filtration, and nuclei were purified by OptiPrep iodixanol gradient centrifugation (25%, 30%, 40%). After centrifugation in a swinging bucket centrifuge (Eppendorf 5810R) for 30 min at 3,000g, nuclei were collected from the 30–40% interface, washed, and assessed by trypan blue staining and microscopy to ensure nuclei integrity. Approximately 15,000 nuclei per sample were processed using the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression platform (10x Genomics).
Tissue preparation and CODEX imaging
Placenta samples for CODEX were OCT-embedded, cryosectioned at 10 µm, mounted on poly-L-lysine-coated slides and stored at −80 °C. On the day of staining, sections were equilibrated, acetone-treated, rehydrated, fixed with 1.6% paraformaldehyde, blocked, and incubated with a barcoded antibody cocktail (200 µl/section) for 3 h at room temperature. Sections were then washed, post-fixed with 4% paraformaldehyde and cold methanol, stabilized using CODEX fixative reagent, and stored in storage buffer at 4 °C (≤2 weeks) before imaging. Multiplex imaging was performed on an Akoya CODEX microfluidic system coupled to an inverted fluorescence microscope using a 7-cycle protocol (including blank cycles for alignment) across 4 channels (DAPI, FITC, Cy3 and Cy5) with a 20×/0.75 NA objective. Images were processed using CODEX Analysis Manager. The antibody panel included Akoya-validated barcoded antibodies and custom-conjugated antibodies generated using Akoya oligo barcodes following the manufacturer’s protocol (Supplementary Table 6). Additional details are provided in the Supplementary Note.
Single-nucleus multiome library construction and sequencing
Single-nucleus Multiome libraries were prepared using the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression kit (10x Genomics) following the manufacturer’s protocol, using one reagent kit per sample. Around 15,000 isolated nuclei per sample were encapsulated into Gel Bead-In Emulsions (GEMs) containing unique cell barcodes, where reverse transcription and transposition occurred, followed by library amplification, and separation of gene expression and chromatin accessibility libraries. Libraries were sequenced on an Illumina NovaSeq 6000 using paired-end reads, with sequencing depth selected on the basis of recommendations from 10x Genomics. On average, each sample yielded approximately 250 million paired-end reads for ATAC and RNA libraries. Raw BCL files were demultiplexed, aligned to the GRCh38 (v.3.0.0) reference genome, and processed for barcode assignment, UMI counting, and quality control using Cell Ranger ARC v.2.0.0 (10x Genomics) (https://support.10xgenomics.com/single-cell-geneexpression/software/pipelines/latest/advanced/references).
Single-nucleus multiome data processing
High-quality nuclei with paired snRNA-seq and snATAC–seq profiles were retained using the following criteria: RNA UMI counts 1,000–50,000; detected genes >400; mitochondrial reads <20%; ATAC fragment counts 1,000–100,000; transcription start site enrichment >1.0; and nucleosome signal <2.0. Doublets were identified and removed using Scrublet62 with prior set to 0.1. After filtering, 191,735 nuclei were retained with paired snATAC- and snRNA-seq data. For snATAC–seq, open-chromatin peaks were called per sample using MACS2 (v.2.2.7)63, and merged into a unified peak set after excluding ENCODE blacklist regions64. Peak-by-cell count matrices were integrated across samples using reciprocal latent semantic indexing (LSI) projection in Signac12. For snRNA-seq, gene expression count matrices were integrated using reciprocal principal components analysis projection in Seurat (v.4)39. Prior to integration, data were normalized, scaled and feature-selected following best practices recommended in Seurat/Signac workflows12.
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