Single-cell RNA-sequencing (scRNA-seq) is an exciting technology that enables useful insights into the genetic patterns of thousands of individual cells. This offers new and vital information for many research applications and disease treatments. To enable users to use this powerful tool most effectively, you need to know how to perform single cell RNA-seq as effective as possible to create reliable and reproducible single cell data.
Fortunately, we recently published our how-to videos and protocol which explain in detailed steps how to perform the Drop-seq protocol. Enquire today to read our protocol in full and to find out more information.
Some of the top tips from the protocol are below:
Accounting for different cell types
Different cell types have different requirements. This affects lysis conditions, single cell suspension methods or even different RNA contents of cells. Those factors and many more will determine if you are able to use a standard Drop-seq protocol or if adjustments are needed to render the workflow suitable for your types of cells or tissues. Especially, finding the optimal lysis conditions for your cells seems to be crucial as some cells are more difficult to lyse then others. Therefore it is important that the instrument will allow for an adaptation of buffers and run parameters to tailor protocols to the needs of your cells.
Bead and Cell counting
Following washing, accurate counting of your beads is required to capture a representative number of cells in a sample. A Fuchs-Rosenthal haemocytometer is recommended due to its increased inlet height of 200 µm. This is easier to use than other cytometers as its increased height enables beads to flow freely through the larger clift. You should also make sure the beads are evenly dispersed to get a true representation of the bead number. Over populated clusters can give a false representation of the bead count.
Additionally, live cells should not be counted – a snapshot of the grid should be taken. Cells move over time and lodge in the grooves and a snapshot ensures the counting is as accurate as possible. It is also important to inject into the grid vertically and to not inject too forcefully to ensure the cells are as evenly distributed as possible.
Handling and Preparation of Beads and cells
Bead handling needs to be done extremely carefully to ensure beads are intact and do not break. It is therefore critical that instructions are followed carefully throughout the Nadia Drop-Seq protocol. Bead handling and mixing should always be done using a pipette, beads should not be vortexed. This will ensure they remain intact and single cell data are as reliable and reproducible as possible.
Cells are fragile and also need to be handled with care. The longer a run takes the more likely it becomes that cells alter their transcriptome or die. Therefore, the preparation is critical. Ensure the Nadia single cell protocol is followed precisely and in a timely manner will help ensure the cells remain stable and intact.
It is also important to ensure the cells are in optimal conditions, which includes being chilled. Chilling cells during cell lysis and mRNA capture reduces batch effects and ensures the data is consistent between runs. The Nadia instrument automatically chills samples to ensure these are kept in an optimal condition.
Another element which is critical to the success of your single cell RNA-Seq experiment is time. Make sure you take measures to minimize the time the processing of your sample takes. The longer the preparation times, the more likely it becomes that cells die or alter their transcriptome. Ensuring that cells are run quickly after single cell suspension have been prepared and are exposed to as little stress as possible.
Furthermore, RNA outside of the cell is very fragile and can easily degrade. Therefore, make sure that ones the lysis of the cells and mRNA capture is completed, emulsions are processed rapidly until the reverse transcription step.
One good tip is to do a few trials runs first before running your actual sample. This ensures you are familiar with the protocol and therefore once running your actual samples you will be quicker and more accurate. This could, for example, include a species mixing experiment using human and mouse cells to control the data quality you are achieving.
Another way to save times is to prepare reaction mixes beforehand wherever possible.
Generally, the PCR cycles should be kept to the lowest number possible. It is widely accepted that the number of PCR cycles used to amplify the cDNA is the major cause of PCR duplicates in sequencing libraries. We typically recommend between 13-16 cycles. However, depending on the cell type or if you are using nuclei this could be higher.
You should keep in mind that the higher the PCR cycle the higher the PCR duplication rate. This can result in a bias towards shorter transcripts and a loss in in read depth.
To find out more about running the Drop-seq protocol on the Nadia Instrument, watch the How to Videos.