Protocols Overview

This section contains the detailed, step-by-step Standard Operating Procedures (SOPs) for the Huang Lab spatial transcriptomics technologies.

RCA Protocols (by Sample Type)

• RCA (Fresh Frozen) — Standard protocol for fresh frozen tissue sections (v5.3)
• RCA (FFPE) — For Formalin-Fixed Paraffin-Embedded samples, with deparaffinization and retrieval (v1.0)

Post-Amplification

• Sequencing & Imaging — In-situ sequencing cycles, morphology staining (DAPI/WGA), and H&E

Reagent Preparation

• Pepsin Solution — HCl dilution and pepsin working solution prep
• WGA-488 Staining — Wheat Germ Agglutinin staining for cell segmentation
• PDMS Fabrication — PDMS substrate for on-slide microfluidic reactions

Workflow Overview

1. Sample Preparation: Cryosectioning (FF) or Deparaffinization (FFPE)
2. Pre-treatment: Fixation, Permeabilization, Blocking
3. Library Construction: Probe hybridization, Ligation, Rolling Circle Amplification
4. Readout: In-situ sequencing (SPRINTseq) or Fluorescence imaging (PRISM)
5. Post-sequencing: Morphology staining (DAPI/WGA), H&E