Where are the validated stopping points when processing FFPE tissues during the Visium HD Spatial Gene Expression assay workflow?

Question: Where are the validated stopping points when processing FFPE tissues during the Visium HD Spatial Gene Expression assay workflow?

Answer: Below are a list of validated stopping points during the Tissue preparation (CG000684) and Library construction (CG000685) steps of the Visium HD assay workflow when processing FFPE tissue samples:

1. During Tissue Slide preparation, after overnight drying, Tissue Slides can then be stored at room temperature or 4°C in a desiccator for up to 6 months.
2. During H&E-stained, archived slide processing, after scraping tissue for RNA quality assessment, the remaining section on the archived slide may be stored in a sealed slide mailer in a desiccator kept in the dark at 4°C for up to one week. Note: The Visium HD FFPE Tissue Preparation Handbook (CG000684) currently indicates two hours, recent experiments have allowed us to extend the storage time. The handbook will be updated in the next revision to contain this info.
3. During Visium HD Spatial Gene Expression library construction:
   • The library construction workflow can occur over two or three days. The first day includes tissue Staining and overnight Hybridization. The second day includes completing the on-slide RNA templated ligation (RTL) leveraging the Visium CytAssist instrument and off-slide library construction steps.
     • If done continuously, the first day is estimated to take ~6 hours (not including imaging time needed), and the second day is estimated to take ~8 hours. This assumes processing two Tissue Slides with one Visium CytAssist instrument.
   • Multiple stopping points have been built into both days of the workflow to maximize flexibility. Please refer to the Visium HD protocols for specifics regarding storage conditions at each of these stopping points.
     1. After completing H&E or IF Staining and coverslip mounting, slides can be stored laying flat in a container at 4°C, in the dark for ≤24h.
     2. After completing H&E or IF imaging coverslip removal and slide drying, slides can be stored in a slide mailer or 50ml centrifuge tube at 4°C, in the dark for ≤2 weeks. Ensure that slides don't come into contact with one another, this may require multiple tubes/mailers to be used.
     3. After completing Post-Ligation Washes, samples can be stored at 4°C for ≤24h.
     4. After completing the second round of Probe Extension, samples can be stored at 4°C for ≤24h.
     5. After completing Pre-Amplification SPRIselect Cleanup, samples can be stored at 4°C for ≤72h or at -20°C for ≤4 weeks.
     6. After completing Sample Index PCR and SPRIselect Cleanup, samples can be stored at -20°C for long-term storage.

Product: Visium HD