Tips for Efficient Droplet Generation of FFPE Samples Using the VITA Platform

Since its introduction in 2023, the VITA single-cell transcriptome platform has empowered research teams to unlock the potential of formalin-fixed paraffin-embedded (FFPE) samples, overcoming long standing barriers in single-cell transcriptome analysis. Leveraging extensive user feedback, VITA’s protocols have been continually optimized to enhance both user experience and data quality. 

In single-cell transcriptome experiments using FFPE samples, droplet generation and cell barcoding are particularly critical, as they can present unique technical challenges due to the nature of FFPE-preserved material. By following these best practices and practical tips, researchers can achieve more consistent results and streamline their workflows, maximizing the value of FFPE samples for high-resolution transcriptomic studies

In this article, we offer essential tips for optimizing droplet generation and barcoding for FFPE samples, aimed at improving the workflow efficiency of your VITA single-cell transcriptome experiments. 

Tip 1: Aspirate the Barcode Beads Carefully and Slowly

When pipetting Barcode Beads, it is crucial to avoid introducing air bubbles. To do this, position the pipette tip at the center of the tube, approximately 2-3 mm above the bottom, and aspirate slowly (as shown in the figure below). Be sure to avoid aspirating the bottommost liquid. 

Tip 2: Mix Single-Nucleus Suspension and Reagents Thoroughly

Before adding sample suspensions and reagents to the chip wells, ensure they are thoroughly mixed. Proper mixing guarantees an even distribution of labeling reagents, leading to consistent and reliable barcoding results.

Tip 3: Pipette Sample Mix and Reagents into the Chip Wells Carefully and Slowly

When adding the sample mix and reagents to the chip wells, align the pipette tip with the center of the well's opening, keeping the tip suspended approximately 1-2 mm above the bottom of the well (as shown in the figure below). Care should be taken to prevent the formation of air bubbles, so reagents should be added to chip wells slowly. 

Tip 4: Attach the Silicone Gasket Correctly

Ensure the silicone gasket is properly attached to the chip to prevent any air leakage. Air leaks can compromise result accuracy, so the gasket must be evenly placed to form a tight, secure seal.

Tip 5: Collect Droplets with Caution

Upon the completion of the droplet generation and barcoding step, carefully collect the liquid from the collection well of the chip. Begin by aspirating and discarding the oil layer from the bottom (as shown in the figure below), then slowly aspirate the droplets along the sidewall of the collection well(s). Transfer the collected droplets into clean PCR tubes for subsequent steps.

By following these tips, researchers can streamline the droplet generation and barcoding processes for FFPE samples on the VITA platform, enhancing both accuracy and efficiency. 

We will be sharing additional tips on VITA single-cell transcriptome experiments to help improve your experimental efficiency and data quality. Stay tuned for more insights on optimizing your experiments!