Single-Cell RNA Sequencing Frequently Asked Questions
Review our Single-Cell RNA-Seq Best Practices for sample submission.
What is Single-Cell RNA-Seq?
Single-Cell RNA-Seq provides transcriptional profiling of thousands of individual cells. This level of throughput analysis enables researchers to understand at the single-cell level what genes are expressed, in what quantities, and how they differ across thousands of cells within a heterogeneous sample.
The workflow for Single-Cell RNA-Seq is outlined below. Isolated cells are frozen by the customer according to Azenta's cryopreservation protocol and shipped overnight. At Azenta's, cell viability is assessed and, if necessary, dead cell removal may be performed (see below for more information). Cells are then processed by the 10x Genomics Chromium™ Controller to create single-cell libraries. The libraries are then pooled and sequenced on the Illumina® platform.
Within the Chromium Controller, cells are loaded onto a microfluidic chip and encapsulated within droplets containing barcoded gel beads and reagents for reverse transcription. Following cell lysis, the beads capture the poly(A) tails of the mRNA molecules. Reverse transcription generates cDNA tagged with a 10x barcode to identify the cell and a unique molecular identifier (UMI) to label the mRNA transcript. The pooled cDNA is amplified in bulk, and Illumina adapters, including a sample index, are added to the fragments to generate sequencing libraries.
Read our article How Single-Cell Sequencing Works for more information.
How does Single-Cell RNA-Seq differ from Standard and Ultra-Low Input RNA-Seq?
Standard RNA-Seq produces a representative snapshot of the transcriptional state averaged across all cells. The caveat with traditional RNA-Seq is the resolution of individual cells and cellular subpopulations are lost. Single-Cell RNA-Seq allows researchers to not only identify cellular subpopulations, but to fully interrogate them at the single-cell level within a heterogeneous sample.
Similar to Standard RNA-Seq, Ultra-Low Input RNA-Seq provides bulk expression analysis of the entire cell population; however, as the name implies, a very limited amount of starting material is used, as low as 10 pg or a few cells. Single-Cell RNA-Seq requires at least 50,000 cells (1 million is recommended) as an input. See below for more information about sample submission guidelines.
Read our article Top 3 Factors to Consider Before Starting a Single-Cell Sequencing Project to learn if single-cell sequencing is the right NGS approach for your experiment.
What is our sample processing workflow?
Our workflow is illustrated here. Viability and cell counts are measured immediately upon sample receipt. To ensure the highest quality data, cells must be processed on the 10x Genomics Chromium system as soon as possible. As such, we developed a predefined workflow when samples do not pass initial QC for viability or cell count.
How do dead cells impact data quality?
Low cell viability causes unreliable cell recovery in the Chromium system and typically leads to missed sequencing targets and suboptimal results. Furthermore, ambient RNA released by dead or apoptotic cells increases background noise, compromising data quality. To maximize the amount of useful sequencing data in single-cell projects, the fraction of dead cells must be minimized. We recommend that cell viability exceeds 90%, with a minimum of 70%. Samples with less than 70% viability may undergo dead cell removal (DCR). A minimum of 106 cells and no previous treatment with magnetic beads is required for DCR. See our workflow for more information.
How does dead cell removal work?
Dead cell removal (DCR) uses magnetic beads conjugated to antibodies against annexin V, a marker for dead and apoptotic cells (see below). The beads capture dead or dying cells, enriching the sample for live cells.
How many cells can be sequenced per sample on the Chromium Controller?
Revealing the full diversity of gene expression across a tissue or cell population often requires analysis of hundreds to thousands of viable cells. With our single-cell workflows, researchers have the flexibility to specify the number of cells to be sequenced, from 500 to 10,000 cells per sample. We typically recommend targeting 3,000 cells per sample for most experiments.
How many reads do I need for my experiment?
The number of reads required depends upon the genome size, the number of known genes, cell type, and transcripts. Generally, we recommend 100,000 reads per cell to maximize the identification of transcripts.
What type of data analysis is available?
We use Cell Ranger software from 10x Genomics for data analysis, which includes the following:
- Read alignment
- Feature-barcode matrix
- Digital gene expression matrix
- Gene expression analysis
- t-SNE projections (shown below)
A sample report is available here (login required).
You can also use the Loupe Cell Browser from 10x Genomics to explore your data, including interactive tools for finding significant genes, identifying cell types, and exploring substructure. More information, tutorials, and demos can be found here.
How is data delivered?
Azenta will recommend the best data delivery option based on the platform and project details. Data delivery options include:
- Secure file transfer protocol (SFTP)
- Customer cloud account
- Hard drive (additional charges may apply)
How will my data be delivered?
- If your results are being shipped to you via hard drive, you can track its shipment using the tracking number provided to you in the delivery email sent from Azenta.
- If your results are being delivered via another data delivery method, such as AWS S3, Aspera, or other cloud-based methods, please refer to the delivery email sent from Azenta for detailed information on the transfer, and consult with your IT department to ensure compliance with your institution’s policies.
- If your results are being sent via a secure File Transfer Protocol (sFTP), please refer to Azenta's sFTP Data Download Guide for instructions on how to download your data and troubleshooting tips.
How do I contact Azenta for a technical consultation about my project?
Azenta's NGS Team is composed of Ph.D. scientists who can help you optimize your project design and provide consultation. See our contact information at the bottom of the page.
What is the price for Single-Cell RNA-Seq?
Please submit a quote request to receiving accurate pricing information, as the cost depends on the details of the project.
How should I prepare and send my samples?
- Sample Type: Frozen cell suspension according to Azenta's supplied protocol, submitted in duplicate. Methanol-fixed cells are also accepted. Tissue dissociation may be available for certain samples (please inquire if interested).
- Recommended Amount: >106 cells in 1 mL
- Minimum Amount: 50,000 cells in 500 µL (Note: 106 cells are required for dead cell removal)
- Buffer: Cryopreservation media (e.g. cell culture media supplemented with 20% FBS and 10% DMSO)
- Tubes/Plates: For projects with <36 samples, prepare samples in clearly labeled microcentrifuge tubes. For larger projects, use 96-well PCR plates.
- Shipment Method: Dry ice overnight. Ship samples directly to our facility. Use the shipping address listed on the order receipt. Note: Azenta has worked with 10x Genomics to develop a proprietary cryopreservation protocol to aid shipment of samples.
What happens if my samples do not meet your starting material requirements?
Please contact firstname.lastname@example.org
Ordering & Processing
How do I request a quote?
For a quick tutorial, watch the video below. Select the Single-Cell Services icon to reach the quote request form.
How do I confirm a quote?
For a quick tutorial, watch the video below.
How do I submit the Azenta sample form (to provide detailed information about my samples)?
For a quick tutorial, watch the video below.>
How can I monitor the progress of my project?
The status of your order, including an estimated date of delivery, can be viewed anytime through your Azenta account. Visit the Order Summary page of your project to find the current order status.
How long do you hold samples?
We hold samples for 1 year after project completion. Please contact us if you would like samples retained for longer or shipped back to you.