Improving Effectiveness for Cell Barcoding of Prokaryotic Samples Using the VITA Platform

The high-throughput single-cell transcriptome products developed by M20 Genomics have gained widespread recognition for providing deeper insights into bacterial transcriptomes at the single-cell resolution. With these innovative products, researchers can explore microbial heterogeneity and gene expression with remarkable precision. In this article, we will introduce some practical tips to streamline workflows and improve experimental outcomes for the critical step of cell barcoding. We hope that these recommendations, drawing on valuable user feedback, can improve the user experience and experimental outcomes.

Tip 1: Optimize cell concentration for barcoding

Before loading the sample into the instrument, it is essential to perform cell counting under a microscope. To do this, prepare a small aliquot of bacterial suspension and stain with propidium iodide (PI). Next, transfer the stained suspension to a glass slide and count the cells under a microscope at 200x magnification. Then adjust the cell concentration to ensure there are 20–100 bacteria per field of view, which is crucial for the subsequent steps.

Tip 2: Avoid bubbles during barcode bead collection

When collecting barcode beads, align the pipette tip with the center bottom of the tube and aspirate slowly to avoid air bubbles. In addition, keep the pipette tip suspended approximately 2–3 mm above the bottom layer (as shown in the figure below), and take care to avoid drawing up the bottommost liquid.

Tip 3: Ensure samples and reagents are well mixed

Be sure to mix the sample suspension and reagents thoroughly before adding them to chip wells. This will ensure reproducibility and reduce variability in subsequent steps.

Tip 4: Handle the samples carefully when loading into chip wells

When loading samples into chip wells, please align the pipette tip with the center of the well opening and suspend it approximately 1–2 mm above the well bottom (as shown in the figure below). Use the first stop of the pipette and dispense slowly to avoid introducing bubbles, which can interfere with droplet generation.

Tip 5: Attach the silicone gasket to the chip

After loading the samples, apply the single-use silicone gasket to cover the chip. Ensure it evenly covers the opening of the chip wells and forms an airtight seal without any gaps.

Tip 6: Use the tilted stand to facilitate droplet collection

When droplet generation is finished, carefully remove the chip and discard the silicone gasket. Then, place the chip on a tilted chip stand for easy droplet collection.

Tip 7: Maximize droplet collection following droplet generation

In the collection well of the chip, droplets form an upper aqueous phase above an oil phase (as shown in the figure below). To maximize the droplet collection, set the pipette to its maximum volume and discard most of the lower oil phase, leaving only a small amount if necessary. Then, slowly aspirate droplets along the sidewall of the chamber and transfer them carefully into collection tubes for subsequent steps. Ensure that the collected droplets are free from excessive oil contamination or air bubbles.

We aim to enhance the accuracy and efficiency of the VITA platform with detailed guidance on cell barcoding steps for prokaryotic samples. By following these guidelines and best practices, users can achieve improved reproducibility and higher data quality for consistent and reliable experimental outcomes.

Stay tuned for future updates, where we'll share additional tips to help you further enhance experimental efficiency and improve data quality when using VITA single-cell transcriptome products.