Optimizing Prokaryotic Sample Preparation: Practical Tips for VITA Single-Microbe Transcriptome

M20 Genomics has introduced VITA Single-Cell Transcriptome Platform for both cultured bacteria and gut microbiome samples, opening new avenues and providing deep insights into prokaryotic research. Drawing from valuable user feedback and experiences, we’ve complied practical insights to help streamline the preprocessing of bacteria samples and formal experimental workflows. We hope these tips will enhance both efficiency and reliability when using VITA products.

Tips for Sample Processing

Tip 1: Use the same method for sample fixation.

There are several effective methods for fixing bacterial samples, each suitable for different experimental conditions and sample characteristics. When selecting a fixation method, it is important to consider these factors. To ensure consistency and comparability of result, it is recommended to use the same fixation method for all samples within a single project.

Tip 2: Minimize the sample loss for single bacterium samples.

If the sample size is small in cultured single-bacterium samples, it is recommended to increase the centrifugation time during fixation to reduce sample loss.

Tip 3: Remove impurities in gut microbiome samples.

For gut microbiome samples like feces, impurities can be removed by centrifugation after fixation. The precipitate represents the impurities, which should be discarded, while the supernatant should be retained for subsequent steps.

Tip 4: Store samples properly if immediate processing is not possible.

If the bacterial sample cannot be processed immediately after fixation, the fixed bacterial suspension can be stored at -80°C. However, it is not recommended to store the sample for more than 6 months.

Tips for Pre-Experiment Procedures

Tip 1: Evaluate fixed bacterial samples with microscope.

For fixed bacterial samples, it is recommended to take a small aliquot of the bacterial suspension for microscopic examination. Briefly, stain the fixed bacteria with PI, then observe them under a microscope at 200x magnification. This microscopy evaluation helps assess cell loss and any morphological changes that may occur in subsequent steps.

Tip 2: Optimize sample dilution for microscopic evaluation.

For the microscopic evaluation mentioned in previous tip, please adjust the dilution of the suspension to achieve 20-100 bacteria per field of view.

Tip 3: Perform bacterial count using microscopic evaluation.

Bacterial counting can be performed using microscopic evaluation. The ideal starting amount should be more than 10 million bacteria, with a minimum of 5 million bacteria required. If the cell concentration is too low, bacteria enrichment is required.

Tip 4: Avoid high bacterial aggregation rate.

Bacterial aggregation can also be assessed through microscopic evaluation. The bacterial suspension should be well-dispersed with minimal impurities, and the aggregation rate should not exceed 20%. An aggregation rate higher than 20% may increase the multiplet rate, which can negatively impact data quality.

Figure. Single bacterial suspension with aggregation rate less than 20%

Tips for Formal Experiment Procedures

Tip 1: Ensure the bacteria suspension remains well-dispersed.

To keep the bacterial suspension well-dispersed, thoroughly pipette the sample after each centrifugation step during the experiment

Tip 2: Utilize ultrasound to reduce cell aggregation.

If bacterial aggregation rate is too high and pipetting alone is ineffective, apply ultrasonic treatment for 10 seconds, no more than three times in total.

Tip 3: Filter the bacterial suspension to minimize cell aggregation.

For bacterial suspension after reverse transcription and adapter ligation, use a 10 µm/5 µm filter for centrifugal filtration if ultrasonic treatment does not effectively reduce cell aggregation.

By implementing these preparation and experimental tips, we aim to help researchers to improve the efficiency and reliability of single-bacterium and gut microbiome sample analysis on the VITA platform.

Stay tuned for future updates, as we'll be sharing additional tips to further improve your experimental efficiency and data quality when using VITA single-cell transcriptome products.