BioMicroCenter:SpTx

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The BioMicro Center supports two complementary approaches to spatial transcriptomics: 10x Visium HD from 10x Genomics for slide-based spatial gene expression, and AVITI24 in situ sequencing from Element Biosciences. The choice of method depends on your tissue type, resolution requirements, and experimental goals. We strongly encourage consultation with BMC staff prior to beginning a spatial experiment.

10X VISIUM HD

Workflow and capture areas for Visium HD Workflow and capture areas for Visium HD

10x Visium HD is a spatial transcriptomics platform that profiles whole-transcriptome gene expression across intact tissue sections at single-cell-scale resolution. Tissue sections are processed through the CytAssist instrument, which transfers RNA complexes onto a capture slide containing a continuous array of 3,000,000 spatially barcoded 2 µm squares covering a 6.5 × 6.5 mm capture area (~0.65 cm²). Two chemistries are available: a probe-based assay for human and mouse (FF, FxF, and FFPE), and a polyA-based assay (Visium HD 3') for fresh frozen tissue from any species. H&E or immunofluorescence (IF) staining can be performed prior to processing for protein co-detection.

ARRAY SPECIFICATIONS Capture area 6.5 × 6.5 mm (~0.65 cm²) per tissue Spots 3,000,000 barcoded squares Spot size 2 µm continuous (no gaps) Default analysis bins 8 µm and 16 µm (SpaceRanger); custom bins supported Tissues per slide 2 Multiomics H&E or IF staining prior to processing; antibody panels (FFPE, human only) CHEMISTRY OPTIONS Visium HD (probe-based) Visium HD 3' (polyA-based) Species Human, Mouse only Any species Tissue formats Fresh Frozen (FF), Fixed Frozen (FxF), FFPE Fresh Frozen (FF) only Notes ~3 probes per gene; exogenous sequences (GFP, reporters) require custom probes. A small fraction of genes are excluded by default due to predicted off-target activity: human excluded gene list (~2.1%), mouse excluded gene list (~1.8%). Species-agnostic; captures polyadenylated transcripts TISSUE PREPARATION REQUIREMENTS Fresh Frozen (FF) Fixed Frozen (FxF) FFPE Section thickness 10–20 µm 10 µm recommended 10–20 µm 5 µm Storage OCT, −80°C −80°C Room temp RNA quality DV200 ≥ 30% recommended; higher scores improve sensitivity Histology Sectioning & H&E performed by the Tang Histology Facility (KI). Coordinate with BMC staff to arrange. For current pricing, see BioMicroCenter:Pricing → Single Cell & Spatial Services → Visium. Sequencing is charged separately; 400–800M reads per tile recommended for Visium HD. SUBMISSION Submit via iLabs (assisted service). Coordinate with BMC staff at least one week in advance. Tissue processing begins at the Tang Histology Facility; please arrange sectioning separately.

10X Visium HD Example of FF data from Visium HD FAQs for Users 1) What tissues are compatible? Visium HD (probe-based) supports human and mouse tissues in FF, FxF, and FFPE formats. Visium HD 3' (polyA) supports fresh frozen tissue from any species. 10x Genomics maintains a list of tested tissues. Additional optimization may be required for tissues with high fat content. Consult BMC staff before starting. 2) How do I prepare and submit tissue? Tissue sectioning and H&E staining are performed by the Hope Babette Tang (1983) Histology Facility at the Koch Institute. Coordinate with both the Histology Facility and BMC staff before your first submission. FF tissue should be embedded in OCT and stored at −80°C; FFPE blocks at room temperature. 3) What is the resolution? Visium HD captures data at 2 µm bin resolution (single-cell scale). SpaceRanger aggregates into 8 µm and 16 µm bins for higher sensitivity by default; custom bin sizes are also supported. Cell segmentation based on H&E imaging is available in the Loupe Browser pipeline. 4) How much sequencing is needed? 400–800M reads per tile is recommended for Visium HD. Sequencing is submitted separately via iLabs. Compatible platforms include NovaSeq SP, NovaSeqX, and Element AVITI. Discuss read depth with BMC staff based on your tissue type and goals. 5) Does Visium HD require permeabilization optimization? No. Visium HD does not require permeabilization time optimization. For fresh frozen tissues new to the platform, a test array is available to verify tissue quality and placement before committing a full experiment. 6) Can I detect proteins alongside gene expression? Yes. H&E or IF staining can be performed prior to CytAssist processing. Antibody panels for protein co-detection (human tissues, FFPE only) are available from 10x Genomics. Discuss your experimental design with BMC staff in advance. 7) What analysis outputs are provided? The BMC delivers FASTQ files, aligned BAM files, the spatial feature-barcode matrix (raw and filtered at 2, 8, and 16 µm bins), and a Loupe Browser file. Downstream informatics support is available through BMC staff. 8) Can I combine Visium HD with single-cell data? Yes. Visium HD data integrates with single-cell or single-nucleus RNA-seq (e.g., 10x Chromium Flex) for cell-type deconvolution. Starting from the same tissue block, nuclei can be isolated for Flex single-cell gene expression. Coordinate with BMC staff if planning a multi-modal experiment. 9) My gene of interest is exogenous (GFP, reporter, viral) or missing from the probe set. What are my options? The probe-based Visium HD assay does not capture exogenous sequences by default. Custom probes can be designed for detection — see 10x Genomics technical note CG000621. Additionally, a small fraction of endogenous genes are excluded from the standard probe sets due to predicted off-target activity: see the human excluded gene list (~2.1% of genes) and mouse excluded gene list (~1.8% of genes). Alternatively, the polyA-based Visium HD 3' assay captures all polyadenylated transcripts and does not require custom probes.

AVITI24 CYTOPROFILING & IN SITU SEQUENCING

Beta Service — Early Access: The AVITI24 Teton CytoProfiling and DISS services at the BioMicro Center are operational but not yet routine. Consultation with BMC staff is required before submitting samples.

The BioMicro Center provides access to the Element Biosciences AVITI24 platform for in situ single-cell multiomics. Using Teton™ CytoProfiling, the instrument co-detects RNA transcripts, proteins (including phospho-proteins), and cell morphology at subcellular resolution — directly in intact cells, without library preparation. Targeted in situ sequencing via Direct In Sample Sequencing (DISS) can be run simultaneously in the same experiment. Because this is an early-access service, results and run parameters are less well-established than our established offerings. Consultation with BMC staff is strongly encouraged before designing experiments.

PLATFORM SPECIFICATIONS Instrument Element Biosciences AVITI24 Technology Teton™ CytoProfiling (ABC Sequencing) Flow cell formats 1-well · 12-well (96-well format) · 48-well (384-well format) Imageable area Up to 10 cm² per flow cell side Max cells per run Up to 1 million adherent cells per flow cell Transcript density ~100–200 transcripts per 100 µm² ASSAY CAPABILITIES Teton CytoProfiling DISS (low output) available DISS (high output) not yet released RNA 350 transcripts panel-based Targeted genes ~50 nt, 1 priming event; CRISPR guides, barcodes, expressed mutations Whole transcriptome polyT prime; 100–300 nt Protein ~18 targets  panels of 24; rabbit monoclonal primary; custom available Morphology Nucleus, actin, membrane (all species) · ER, Golgi, mitochondria (human only) Panels / probes MAPK Cell Cycle · MAPK Apoptosis · Immuno-Oncology · Neuroscience mix up to 3 panels per run; consult staff for latest User-designed probes User-designed or polyT probe Teton CytoProfiling and DISS (low output) can be run simultaneously in the same experiment. Custom antibody add-ons require unconjugated rabbit monoclonal primaries. Consult BMC staff for current options. SAMPLE REQUIREMENTS Supported Monolayer cell culture (adherent); pelleted cell suspension (spun down and resuspended) Not yet supported Complex 3D culture, organoids, tissue sections — contact BMC to discuss future availability Cell density Consult BMC staff; optimized per cell type Species Human (all current fixed panels); other species — consult staff regarding panel compatibility SUBMISSION Submission via iLabs is not yet available. Contact BMC staff directly to discuss your experiment before scheduling. Consultation is required for all AVITI24 Teton and DISS submissions.

AVITI24 Teton CytoProfiling at BMC Cytoprofiling of cardiomyocytes. Collaboration with the Boyer Lab. About the Service The AVITI24 at BMC supports Teton™ CytoProfiling — an in situ multiomics approach that co-detects RNA transcripts, proteins (including phospho-proteins), and cell morphology directly inside intact cells on the flow cell. Each run cycles through Cell Painting (morphology imaging), antibody scoring, and in situ sequencing rounds without any library preparation. A full run takes 24–48 hours end-to-end with approximately 45 minutes of hands-on time, capturing ~100–200 transcripts per 100 µm² at subcellular resolution. The platform is designed for cells in monolayer culture or spun-down suspension. It profiles up to 350 RNA targets, ~18 protein targets, and 6 morphology channels simultaneously in a single experiment — giving a co-registered multiomic readout per cell that would otherwise require separate assays on separate instruments. Targeted in situ sequencing (DISS, low output) can be layered onto any Teton run. This uses a user-designed probe (~50 nt, 1 priming event) to detect specific sequences in situ — useful for CRISPR guide detection, barcode readout, or expressed mutation calling. Note that reads are too short for TCR/BCR sequencing; consult BMC staff if your application involves immune repertoire. Available Assays BMC supports all four currently available fixed Teton panels (MAPK Cell Cycle, MAPK Apoptosis, Immuno-Oncology, Neuroscience). Up to three panels can be combined per run, giving up to 350 RNA targets and ~18 usable protein targets in a single experiment. Custom antibody add-ons are possible using unconjugated rabbit monoclonal primary antibodies — no conjugation or barcoding required. Discuss panel selection with BMC staff; reagent availability and lead time vary. Data Outputs The BMC provides core analysis outputs including: Cell-by-gene expression matrix (350 transcript targets per panel) Cell-by-protein parameter data (~18 antibody targets) Morphology feature data per cell (nucleus, actin, membrane; ER, Golgi, mitochondria for human) Spatial maps with morphology channel overlays ElemBio Catalyst / CytoCanvas-compatible files for interactive visualization and downstream analysis Additional informatics support is available through BMC staff. Pricing See BioMicroCenter:Pricing → Single Cell & Spatial Services → AVITI24 for current pricing. Sequencing-only runs on the AVITI24 are charged at standard AVITI24 sequencing rates. Service Status This service is in beta. The instrument is operational and BMC has completed initial runs, but experimental parameters are still being validated. We welcome early users willing to work collaboratively with BMC staff to establish protocols.

SINGULAR G4X IN SITU SPATIAL SEQUENCING

Beta Service — Early Access: The Singular G4X at the BioMicro Center is newly installed and not yet in routine service. Consultation with BMC staff is required before submitting samples.

The BioMicro Center provides access to the Singular Genomics G4X, an upgrade to the G4 sequencer that adds in situ spatial multiomics capability. The G4X simultaneously profiles targeted RNA transcripts, proteins, and fluorescent H&E (fH&E) directly in intact FFPE tissue sections at subcellular resolution. Runs are organized around individual flow cells; BMC will typically run one flow cell at a time. Because this is an early-access service, results and run parameters are less well-established than our routine offerings. Consultation with BMC staff is strongly encouraged before designing experiments.

FLOW CELL OPTIONS (per slide) Flow cell Lanes ROI size Samples Imaging area Run time X2 2 10 × 10 mm 10 10 cm² ~29 hrs X4 4 4.5 × 4.5 mm 32 6.5 cm² ~24 hrs Run times do not include analysis time (~1 day per flow cell after imaging). Consult BMC staff to select the appropriate flow cell for your project. PLATFORM SPECIFICATIONS Instrument Singular Genomics G4X Spatial modalities Targeted RNA (in situ), targeted protein, fluorescent H&E (fH&E) RNA plex Up to 500-plex per run Protein plex Up to 18-plex per run Tissue format FFPE (primary); other formats — consult BMC staff Sample-to-answer ~5 days For current pricing, see BioMicroCenter:Pricing. Details to be added as the service matures. SUBMISSION Submission via iLabs is not yet available. Contact BMC staff directly to discuss your experiment before scheduling. Consultation is required for all G4X submissions.

Singular G4X at BMC The Singular G4X enables in situ spatial multiomics by co-detecting RNA transcripts, targeted proteins, and fluorescent H&E simultaneously in the same FFPE tissue section at subcellular resolution. Tissue sections are transferred to flow cells using Singular's TissuStamp™ system, and the instrument images the full flow cell in a single day. BMC will typically run one flow cell at a time, with flow cell choice (X2 or X4) determining sample capacity and ROI size. The platform uses application-specific RNA panels (290–500 plex depending on tissue type) and a protein panel (up to 18-plex). Panels can be customized with add-ons of up to 50 genes, new 150-gene modules, or fully custom 500-gene panels. Current off-the-shelf panels include Lung, Colon, Breast, Kidney, Pancreas, Skin, and Pan-Mouse. See the Singular content page for the full panel list and gene targets. For more detail on chemistry, workflow, and analysis, see the G4X Platform Ecosystem and G4X in situ multiomics pages from Singular Genomics. Service Status This service is in beta. The G4X is newly installed and BMC is establishing protocols and validated run parameters. We welcome early users willing to work collaboratively with BMC staff. Contact biomicro@mit.edu to discuss your project.