Table 2.
Protocols used in different spatial transcriptomic methods (part 2)
| Salus | DynaSpatial | Slide-seq V2/Curio Bio | Visium(probe-based) | |
|---|---|---|---|---|
| Tissue optimization (preexperiment) | 1 Tissue fixation | 1 Tissue fixation | 1 Collect tissue sections | |
| 2 Tissue H&E staining | 2 Tissue H&E staining | 2 RNA isolation | ||
| 3 Tissue brightfield imaging | 3 Tissue brightfield imaging | 3 RNA QC by RNA integrity number (RIN ≥ 4) | ||
| 4 Permeabilization time course | 4 Permeabilization time course | none | ||
| 5 Cys cDNA synthesis | 5 Cys cDNA synthesis | |||
| 6 Tissue removal | 6 Tissue removal | |||
| 7 Slide Cy3 imaging | 7 Slide Cy3 imaging | |||
| Permeabilization time | Brain: 6 min | Brain: 10 min | no permeabilization required | no permeabilization required |
| Embryo: 6 min | Embryo: 10 min | |||
| Spatial expression (formal-experiment) | 1 Cryosection on the Salus chip | 1 Cryosection on the DynaSpatial slide | 1 Cryosection on bead arrays | 1 Cryosection on the glass slide |
| 2 Fixation: methanol for 30 min | 2 Fixation: methanol for 30 min | 2 mRNA hybridization for 15 min | 2 Fixation: methanol for 40 min | |
| 3 Tissue H&E staining | 3 Tissue H&E staining | 3 Reverse transcription | 3 Tissue H&E staining | |
| 4 Tissue brightfield imaging | 4 Tissue brightfield imaging | 4 cDNA release | 4 Tissue brightfield imaging | |
| 5 Tissue permeabilization | 5 Tissue permeabilization | 5 cDNA amplification | 5 Probe hybridization and ligation | |
| 6 Reverse transcription | 6 Reverse transcription | 6 cDNA cleanup and cDNA QC | 6 Probe release in CytAssist and probe extension | |
| 7 cDNA release | 7 cDNA release | 7 Preamplification and cleanup | ||
| 8 cDNA amplification | 8 cDNA preamplification | |||
| 9 cDNA cleanup and cDNA QC | 9 cDNA cycle number determination by quantitative PCR | |||
| 10 cDNA amplification | ||||
| 11 cDNA cleanup and cDNA QC | ||||
| H&E: same tissue section | H&E: same tissue section | H&E: same tissue section | ||
| Library construction | cDNA was fragmented and amplified, then the products were filtered twice. | 1 Cycle number determination by quantitative PCR | ||
| To construct the sequencing library, cDNA was fragmented, end-repaired and A-tailed. Then the adapter was ligated so the dual index PCR could amplify and distinguish these samples by different index sequences. The distribution of the main peak was between 200 and 600 bp and the libraries were sequenced in the same sequencing run. | 2 Sample index PCR and cleanup (0.85×) | |||