Illuminating helpful insights into thousands of individual cells’ genetic patterns, single-cell RNA-sequencing (scRNA-seq) is a fascinating technology which offers crucial, cutting-edge information for several disease treatments and research applications. If users are to get the most out of this powerful tool, they must have the knowledge to perform single-cell RNA-seq as effectively as possible. This creates both reliable and reproducible single-cell data.
Here are some top tips on how to perform the Drop-seq protocol.
Accounting for Different Cell Types
There are different requirements for different cell types, which affect lysis conditions as well as single cell suspension methods and even different RNA contents of cells. Whether or not you can utilize a standard Drop-seq protocol is determined by these and several other factors. Alternatively, rendering the workflow suitable for your types of cells or tissues may require adjustments.
As some cells can be more difficult to lyse than others, it is of particular importance to find the optimal lysis conditions for your cells. Consequently, in order to adjust protocols to your cells’ needs, it is vital that the instrument allows for both buffers and run parameters to be adjusted.
Bead and Cell Counting
After you have washed your beads, they must be accurately counted in order to capture a representative number of cells in a sample. Consequent upon its greater inlet height of 200 µm – which allows for the free flow of beads through a larger cleft – it is recommended to use a Fuchs-Rosenthal haemocytometer as it is easier to use than other cytometers.
Also, in order to ascertain an accurate representation of the bead number, it is necessary to check that the beads are evenly dispersed. This prevents over-populated clusters giving false representations of the bead count.
Over time, cells shift and lodge in the grooves. As a result, instead of counting live cells, you should take a snapshot of the grid, as this ensure the count is as accurate as possible. You must also inject vertically into the grid without too much force, as this guarantees the most even distribution of cells.
Handling and Preparation of Beads and Cells
To ensure the structural integrity of beads, they should be handled with extreme care. Consequently, instructions must be meticulously followed for the duration of the Nadia Drop-seq protocol. Pipettes should be used whenever you are mixing or handling beads, and to make sure they remain intact, beads should not be vortexed. This ensures the greatest possible reliability and reproducibility of single cell data.
You must similarly exercise caution in the handling of cells, as they are also fragile. As cells become more likely to change their transcriptome or die the longer a run lasts, the preparation is critical. Make sure you abide by the Nadia single cell protocol strictly and efficiently. This helps cells to remain both stable and intact.
As cells must remain in optimal conditions, they need to be chilled. If cells are chilled during lysis and mRNA capture, batch effects are reduced and the consistency of data between runs is ensured. In order to guarantee that samples are kept in optimal conditions, the Nadia instrument automatically chills them.
Time is another crucial factor in the success of a single cell RNA-seq experiment. To minimize the processing time of your sample, you must take measurements. Cells are more likely to either die or alter their transcriptome the longer you take to prepare the experiment. Make sure that cells are run swiftly after single cell suspension has been prepared. It is also important that they are exposed to the least possible degree of stress.
Additionally, outside of the cell, RNA is both extremely fragile and easily degradable. It is important to ensure, therefore, that upon the completion of cell lysis and mRNA capture, emulsions are processed very quickly up to the reverse transcription step.
It is advisable to undertake some trial runs prior to the running of your actual sample, to establish your familiarity with the protocol. As a result, you will be more accurate and efficient when running the actual samples. As an example, you may consider undertaking a species mixing experiment with human and mouse cells. This can help to control the data quality you achieve.
If possible, prepare reaction mixes prior to the experiment in order to save time.
In general, PCR cycles must be limited to the lowest possible number. Scholarly consensus accepts that the major cause of PCR duplicates in sequencing libraries is a high number of PCR cycles used to amplify the cDNA.
It is recommended to use somewhere in the region of 13-16 cycles. Yet, this could be higher – it depends on the cell type and whether you are using nuclei.
Remember: a higher PCR cycle means a greater PCR duplication rate, which can result in both loss in read depth and a bias towards shorter transcripts.
About Dolomite Bio
Dolomite Bio creates innovative products for high throughput single cell research including the Nadia Instrument and the Nadia Innovate. By encapsulating single cells in microfluidic droplets, our products enable rapid analysis of thousands or millions of individual cells and their biological products.
Key benefits of Dolomite Bio systems:
- Flexible: The systems enable full control of pressures, flow rates and temperatures
- Compatible with a range of applications: Not limited to a specific protocol
- Productised: Complete off-the-shelf, easy to use systems
- High precision with reproducible results: Monodisperse droplets with low doublet rate of cells and beads
Our products are complete systems enable scientists to generate thousands of single cell libraries in just a few minutes.
Our in-house team of biologists and worldwide network of local specialists work with you to provide advice for your application, product demonstrations, installation, training and support.