What is scRNA-seq, and how does it differ from bulk RNA-seq?
Single cell RNA-seq is a genomic approach that enables the profiling of gene expression at the single-cell level. Unlike bulk RNA-seq, which averages gene expression across a population of cells, scRNA-seq provides insights into cell-to-cell heterogeneity.
Why is scRNA-seq important for biomedical research?
Single cell RNA-seq allows us to study cellular responses, identify rare cell types, explore cell states, and uncover novel biomarkers. It has revolutionized our understanding of cell biology and disease mechanisms.
What are the key steps in a scRNA-seq experiment?
The typical workflow includes:
- Isolation of single cells/nuclei and 10x Genomics library preparation
- Whether the starting material is tissue or cells, samples are quality checked and prepared for the downstream processing by SCD.
- Read more about the minimal sample requirements or book a meeting with our specialist to discuss your projects specifics.
- Sequencing
- Sequencing service is provided as part of your single cell experiment with us or as a standalone service if you have your libraries ready. Read more about our sequencing service or reach out to us.
- Bioinformatic data analysis
- Our bioinformatic team performs the necessary QC on your data and provides a report as part of our standard service
- If you need assistance with further data processing, our Data consultancy team is at your disposal.
How do I choose the right protocol for my scRNA-seq study?
Consider factors such as cell type(s) of interest, sample size, desired sensitivity and cost. For example, if you have a rather large sample size or need to profile tens of thousands of cells to answer your research question, 10x Genomics platform is a great choice. If you have a particular interest in immune cell profiling, 5’ Immune profiling can be leveraged to identify T and B cell populations. If you have a large sample size or formaldehyde fixed cells, Fixed RNA profiling (Flex) could be a cost effective probe based method of choice. If you work with limited samples and wish to profile hundreds of cells, our SORT-seq or full transcriptome VASA-seq services might be suitable. For more information and help with planning the experimental setup, read more about our services or book a meeting with our specialists.
What 10x Genomics services does SCD offer?
We currently offer:
- 3′ Single Cell Gene expression profiling (Next GEM v3.1 and GEM-X v4)
- 5′ Single Cell Immune Profiling with TCR and BCR V(D)J analysis (Next GEM v2 and GEM-X v3)
- Single Cell ATAC-seq (Next GEM v2)
- Single Cell Multiome ATAC + Gene Expression (Next GEM v1)
What are the add-ons and customization options for the 3′ Gene expression protocol?
The 3′ Gene expression protocol can be done on whole cells or nuclei. Whole cell approach offers Feature Barcoding add-on such as Cell surface protein (e.g CITE-seq) detection. Custom R&D add-ons are also possible after consultation with R&D. For more information contact us to discuss the possibilities.
What are the add-ons and customization options for the 5′ Immune profiling protocol?
The 5′ Immune profiling protocol includes optional BCR and/or TCR libraries. If the protocol is applied to whole cells Feature Barcoding add-on such as Cell surface protein (e.g CITE-seq) detection as also possible. For R&D customizations reach out to us!
What are the add-ons and customization options for the Fixed RNA profiling (Flex) protocol?
The Flex protocol can be done on tissues, whole cells or nuclei. Whole cell approach offers Feature Barcoding add-on such as Cell surface protein (e.g CITE-seq) detection. Custom R&D add-ons are also possible after consultation with R&D. For more information contact us to discuss the possibilities.
What are the differences between 3’ and 5’ scRNA-seq assays?
While both assays use polydT primers for RT, they differ in the location of barcoding and the end of the transcript they capture. Researchers choose between 3’ and 5’ assays based on their specific biological questions and experimental goals.
3’ scRNA-seq (Single Cell 3’ Gene Expression):
- Capture End: The 3’ assay captures the 3’ end of the polyadenylated transcript.
- PolydT Primer: It uses a polydT primer for reverse transcription (RT), with the polydT sequence located on the gel bead oligo.
- Barcoding: The 10x Barcode is adjacent to the polyA tail on the 3’ end of the transcript.
- Final Library: The resulting library represents the 3’ end of the transcript.
- Applications: Commonly used for gene expression profiling and identifying cell types.
5’ scRNA-seq (Single Cell 5’ Gene Expression):
- Capture End: The 5’ assay captures the 5’ end of the transcripts.
- PolydT Primer: It also uses a polydT primer for RT, but the sequencing barcodes are not adjacent to the polydT primer.
- Barcoding Location: The sequencing barcodes are at the 5’ end of the transcripts.
- Final Library: The resulting library represents the 5’ end of the transcript.
- Applications: Useful for studying transcription start sites, alternative splicing, and isoform diversity.
What is Fixed RNA profiling (Flex)?
Flex is the latest 10x Genomics Single Cell Gene Expression solution which empowers comprehensive profiling of the transcriptome in single cells. By utilizing a probe-based approach, it captures gene expression data from Additionally, the incorporation of Feature Barcode technology enables simultaneous profiling of cell surface protein expression, providing multidimensional insights into complex biological processes. Furthermore, this assay supports sample multiplexing, enhancing throughput and workflow efficiency.
When to choose Flex over 3′ Gene expression?
Flex is preferred over the 3′ Gene expression protocol in certain cases, such as:
- When a large number of samples need to be processed – one Flex reaction can contain up to 16 samples increasing the cost efficiency per sample
- When formaldehyde fixation provides advantages such as in time course experiments
- Samples are sensitive to cryopreservation – FACS sorted, or chemically treated samples can be of reduced viability and cryopreservation is not ideal option. Samples can alternatively be formaldehyde fixed formaldehyde-fixed (with Chromium sample preparation kit) and shipped to SCD.
- Limited tissue amount – Flex can work with as little as 10 mg of tissue (depending on tissue type)
When is it not recommended to choose Flex over 3′ Gene expression?
Unlike reverse transcription based 3′ Gene expression solution, Flex is a probe-based method. This means that samples are hybridized with a pre-defined set of probes, currently only available for human and mouse transcriptome and targeting the canonical coding transcriptome. Therefore, different model organisms or interest in studying alternative transcripts might be better suited for 3′ or 5′ Gene expression. The full set of Flex probes and targeted genes is available at the official 10x Genomics webpage.
How many cells should I aim for per sample for 10x Genomics experiment?
That depends on your biological question and sample type. Typically, our customers target between 3,000 and 10,000 cells per sample. With the new GEM-X technology it is possible to profile up to 20,000 cells per sample. Schedule a call with one of our specialists to discuss your specific project.
How many reads per cell should I aim for?
That depends on your biological question. Typically, we recommend a sequencing depth between 30,000 and 70,000 reads per cell for 10x Genomics projects. Schedule a call with one of our specialists to discuss your options.
Can I enrich my sample with FACS sorting?
Yes, before you send us your sample, you can perform a FACS sort to enrich your cell type of interest. Please remember minimum cell and viability requirements. You can find out more about sample requirements here.
What are the sample requirements for the 3′ Gene expression and 5′ Immune profiling protocols?
The most important QC metrics for both the 3′ Gene expression and 5′ Immune profiling protocols are the cell number, viability and debris and/or cell clumping.
- The minimum required cell number is at 500,000 cells, however, lower numbers can be processed after discussing the setup with SCD.
- Optimal cell viability is at >90%, but it is possible to run samples with >70% – with caution. When determining cell viability it is important to perform the assessment after the (cryo)preservation of cells, with a reliable method, preferably AO/PI staining or other fluorescent dyes.
- Ideally, samples will have minimal cell debris and/or clumping. SCD performs filtering as part of our standard protocol to minimize the presence of either.
Read more about sample requirements or schedule a call with us.
What are the sample requirements for the Fixed RNA profiling (Flex) protocol?
For cells, at least 300,000 cells or 500,000 nuclei are required for fixation. For snap-frozen tissues, generally at least 20 mg of tissue is required, but it also depends on the tissue type. Prior to fixation it is highly recommended to assess the samples viability (when applicable), ideally >80%. Like the other protocols, the presence of debris and clumping should be considered, but not crucial due to extensive washes after fixation and hybridization steps of the protocol.
Read more about sample requirements or schedule a call with us.
Can I submit tissues for the 3′ Gene expression or 5’ Immune profiling?
For the standard throughput 3′ Gene expression or 5’ Immune profiling experiments without Feature Barcoding, tissues will be subjected to nuclei extraction during the processing. Due to damage caused to cells by snap-freezing, it is not possible to process samples as whole cells. SCD has extensive experience with a variety of tissue types, for more information contact us to discuss the possibilities.
What are the sample preservation methods for the 3′ Gene expression and 5′ Immune profiling protocols?
For both the 3′ Gene expression and 5′ Immune profiling protocols, samples can be cryopreserved in a freezing medium of your choice (usually consisting of 50% FBS, 40% basal growth media, and 10% DMSO). Cryopreserved cells can be shipped on dry ice. Alternatively, fresh cells can be submitted on ice when cryopreservation is not suitable. When delivering fresh samples to SCD, it is necessary to discuss with our team prior and arrange the delivery. Methanol fixation can be an alternative option, albeit it is not compatible with the 5′ Immune profiling protocol.
What are the sample preservation methods for the Fixed RNA profiling (Flex) protocol?
The Fixed RNA profiling (Flex) protocol offers multiple sample preservation methods.
- Fresh cells can be submitted on ice if an arrangement has been previously made with SCD.
- Cryopreserved cells and snap frozen tissues can be shipped on dry ice, for more information take a look at our sample shipping
- Formaldehyde-fixed (with Chromium sample preparation kit) cells can be submitted after formaldehyde fixation and preservation according to the protocol. We strongly advise performing a viability assessment prior to fixation for formaldehyde-fixed samples and provide the viability information along with the sample submission. For accurate viability assessment we recommended using an automated cell counter with fluorescent stains or alternatively Trypan blue staining.
Read more about sample requirements or schedule a call with us.
What happens if the samples do not pass the quality control (QC)?
If the samples do not pass the QC, the experiment is paused (when applicable) or aborted. 3’ Gene expression and 5’ Immune profiling experiment success relies on quality of fresh sample, primarily cells viability (or nuclear integrity). In case of failed QC, the samples cannot be stored and experiment is aborted. Formaldehyde fixed samples for Fixed RNA profiling (Flex) protocol can be safely stored for 6 months, therefore in case of failed QC we preserve your samples until next steps are discussed.
Due to the possibility of samples not meeting the minimum quality requirements, we recommend setting up a QC-only experiment with us. Reach our to our specialists to help you with a setup.
Can I perform a QC experiment for my samples?
Yes, we recommend performing a test QC experiment with spare samples before submitting your samples of interest to SCD. We can help you set up the pilot conditions and let you know what to look for!
What is the DV200 score, and how is it related to FFPE samples?
The DV200 score is a quality metric used to quantify the percentage of RNA fragments in a sample that are longer than 200 nucleotides (nt). In the context of FFPE (formalin-fixed paraffin-embedded) samples, the DV200 score is particularly relevant for assessing the quality of RNA extracted from FFPE samples. A higher DV200 score indicates a larger proportion of intact RNA, which is crucial for accurate downstream analysis. If the DV200 score is low (e.g., below 50%), it may be necessary to consider alternative approaches to single cell RNA-seq. When sending FFPE samples to SCD, make sure to include additional scrolls for DV200 analysis. Read more about FFPE sample requirements or book a meeting with our specialist.