Spatial transcriptomics sample preparation, tissue optimization, library preparation, and quality control workflow

Capture region- or cell-scale transcripts while preserving tissue spatial location and morphological information, completing tissue quality assessment, section optimization, spatial capture, library construction, and pre-sequencing quality control.

Experimental Objectives

To capture region- or cell-scale transcripts while preserving tissue spatial location and morphological information, completing tissue quality assessment, section optimization, spatial capture, library construction, and pre-sequencing quality control.

Scope of Application and Experimental Requirements

  • Sample: Fresh frozen tissue
  • Sample: FFPE tissue validated by the platform
  • Sample: Organoids and 3D cultured samples
  • Sample: Tissue sections with pre-assessed RNA quality
  • Species: Human, mouse, rat, and other species with high-quality reference genomes and annotations
  • Difficulty and Time: Advanced, approximately 3–7 days; sequencing and bioinformatics analysis extra
  • Safety: Human and animal-derived tissues require corresponding approvals; sectioning, fixation, and staining should follow sample risk and chemical regulations; pre- and post-amplification partitioning and RNase and cross-sample control should be performed.

Experimental Principle

Place qualified tissue sections on a capture area with spatial barcodes, then release RNA through fixation, staining, imaging, and appropriate permeabilization; after transcripts bind to positional barcodes, reverse transcription, amplification, and library construction are completed, and sequencing data is registered with tissue images.

Standard Workflow

  1. Develop plans for tissue collection, ischemia time, embedding, and batch balancing
  2. Assess tissue morphology, RNA quality, and section integrity
  3. Optimize section thickness, adhesion, fixation, staining, and permeabilization conditions
  4. Complete tissue sectioning and high-quality imaging in the capture area
  5. Perform in situ capture, reverse transcription, and spatial barcoding
  6. Recover and amplify cDNA, and complete product quality inspection
  7. Construct, purify, quantify, and verify spatial expression libraries
  8. After sequencing, complete image registration, quality filtering, spatial clustering, and marker validation

Equipment, Reagents, and Consumables

  • Equipment: Cryostat or paraffin sectioning equipment, microscopic imaging system, temperature control and humidity chamber equipment, PCR machine, and library quality analysis equipment
  • Reagents: Tissue embedding and section processing reagents, fixation, staining, and permeabilization systems, spatial capture and reverse transcription reagents, library amplification, purification, and quantification reagents
  • Consumables: Spatial capture slides or chips, RNase-free low-retention tubes and filtered pipette tips, sectioning consumables and coverslips, PCR plates and sealing films

Essential Controls and QC

  • RNA quality assessment of adjacent sections
  • Tissue morphology reference section
  • Permeabilization time gradient or optimized section
  • Tissue-free background area
  • Library negative and batch reference controls
  • Complete tissue structure with sufficient target region coverage; RNA and morphological quality meeting platform requirements; controlled background signal; qualified cDNA and library concentration and fragment distribution; effective spot count, reads, gene count, and intra-tissue to extra-tissue signal ratio meeting project standards.

Key Parameters and Result Interpretation

Tissue collection ischemia time, tissue preservation, section thickness, RNA integrity, permeabilization time, imaging quality, capture area coverage, amplification cycles, and sequencing depth collectively determine spatial resolution and gene detection.

Spatial expression reflects mixed cellular signals within the measured area, and resolution and tissue thickness can affect cell type interpretation. Pathological annotation, single-cell references, protein imaging, and independent sample validation should be combined to verify key spatial patterns.

Common Problems and Troubleshooting

Optimize slide handling and fixation for tissue detachment; shorten ischemia time and improve preservation for low RNA quality; adjust permeabilization and washing for high background outside the tissue; verify section quality, capture area, and amplification conditions for low library complexity; unify imaging and tissue boundary annotation for registration deviation.

Product BOM Entry

Submit product and technical inquiries for this experiment

Research Use Statement: This workflow is for research experiment design and product selection only; specific parameters, compatibility, specifications, prices, and delivery are subject to project evaluation and formal quotation.