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Protocols

Step-by-step Standard Operating Procedures (SOPs) for the Huang Lab spatial transcriptomics workflow.

New to spatial transcriptomics?

Start with the Technologies Overview to understand the complete workflow before diving into protocols.

Quick-Start: Complete Workflow Examples

The most common workflow. Direct RNA ligation on fresh frozen cryosections, PRISM single-round imaging readout.

Step Protocol Time
1. Pre-treatment Fresh Frozen ~1.5 h
2. Library construction RCA Protocol (Hybridization → Ligation → RCA → Post-fix) ~18 h (overnight RCA)
3. Readout PRISM Imaging (Fluorescent probe hybridization + imaging) ~2 h
4. Morphology DAPI + WGA (optional) ~0.5 h
Total ~22 h (1 overnight)

For archival FFPE tissue. Adds deparaffinization, RNA retrieval, and NaBH₄ autofluorescence quenching.

Step Protocol Time
1. Pre-treatment FFPE (Baking + deparaffinization + retrieval + NaBH₄) ~20 h (overnight baking)
2. Library construction RCA Protocol + Autofluorescence Quenching (likely needed) ~18 h (overnight RCA)
3. Readout PRISM Imaging ~2 h
4. Morphology DAPI + H&E (optional) ~1 h
Total ~41 h (2 overnights)

Workflow Overview

graph TD
    A["<b>1. Sample Pre-treatment</b><br/><small>FF or FFPE</small>"] --> B["<b>2. RCA</b> (unified)<br/><small>Hybridization → Ligation →<br/>RCA → Post-fix</small>"]
    B --> C{"Autofluorescence<br/>quenching?"}
    C -->|"Optional<br/>(sample-dependent)"| D["Autofluorescence<br/>quench"]
    C -->|No| E
    D --> E["<b>3. Signal Readout</b>"]
    E --> E1["PRISM<br/><small>Single-round imaging</small>"]
    E --> E2["SPRINTseq<br/><small>In-situ sequencing</small>"]
    E1 --> F["<b>4. Morphology</b>"]
    E2 --> F
    F --> F1["DAPI<br/><small>Nuclear</small>"]
    F --> F2["WGA<br/><small>Cell membrane</small>"]
    F --> F3["H&E<br/><small>Histology</small>"]
    F --> F4["IF<br/><small>Protein</small>"]

    style A fill:#E7EDF1,stroke:#5B7488
    style B fill:#E9EEE6,stroke:#5F7A57
    style E1 fill:#E9EEE6,stroke:#5F7A57
    style E2 fill:#E7EDF1,stroke:#5B7488
    style F1 fill:#F3E6DE,stroke:#A8432A
    style F2 fill:#F3E6DE,stroke:#A8432A
    style F3 fill:#F3E6DE,stroke:#A8432A
    style F4 fill:#F5ECDA,stroke:#B5832F

1. Sample Pre-treatment

Choose based on your sample type. Both protocols end at the same point (tissue ready for probe hybridization).

Protocol Sample Type Description
Fresh Frozen Cryosections (v5.3) Fixation, permeabilization, dehydration
FFPE FFPE (v1.0) Deparaffinization, RNA retrieval, rehydration, NaBH₄ quenching, digestion

2. Library Construction

Protocol Description
Strategies Overview Comparison of ligation strategies: direct RNA, chimeric padlock, cDNA, iLock. Read this first to choose your approach.
RCA Protocol Unified protocol: Hybridization → Ligation → RCA → Post-fix. Autofluorescence quenching is a separate optional step.

3. Signal Readout

Choose based on your readout technology:

Protocol Technology Description
PRISM Imaging PRISM Fluorescent probe hybridization + single-round multi-channel imaging
SPRINTseq Sequencing SPRINTseq Multi-cycle in-situ sequencing-by-synthesis in flow cell

4. Morphology (Post-Readout)

Performed after PRISM or SPRINTseq readout. Each is independent and optional:

Protocol Target Description
DAPI Cell nuclei Nuclear staining for segmentation
Cell Membrane (WGA) Cell membrane WGA-Alexa 488 for cell boundary segmentation
H&E Tissue histology Standard hematoxylin & eosin for morphological context
Immunofluorescence Protein (multi-modal) Antibody-based protein detection; provides protein-level spatial data complementing RNA

Materials & Reagent Preparation

Stored stocks and fabricated materials — prepare these ahead of a run. Anything made fresh at the bench stays inline in the protocol that uses it.

  • Pepsin Solution -- HCl dilution and pepsin storage/working solution prep
  • DAPI Stock -- 5 mg/ml stock and serial dilution to the 10 ng/ml working concentration
  • WGA-488 Stock -- 2 mg/ml stock, storage, and pre-use clarification
  • PDMS Chamber Fabrication -- casting and punching the 100 µl on-slide reaction chamber

Background

Reference

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