Overview
Illumina Single Cell 3' RNA Prep kits make single-cell RNA sequencing (scRNA-Seq) accessible for more labs with an easy, scalable, and microfluidics-free workflow. Researchers can undertake larger and more varied multiomics studies without the budget or technology constraints of other single-cell approaches.
Scalable and flexible solution
Illumina Single Cell 3' RNA Prep can easily scale with a wide processing range from hundreds to hundreds of thousands of cells. This increased cell throughput can reveal rare cell types. The assay is also flexible to run one sample or up to 96 samples in parallel with sample multiplexing.
High-quality performance
Illumina Single Cell 3' RNA Prep kits offer high transcript and gene sensitivity with the high-quality data you expect from Illumina. The gentle isolation technique helps detect fragile cells that are often missed by other methods. The assay is compatible with custom single-cell measurements for expanded multiomic applications.
Accessible workflow
Illumina Single Cell 3' RNA Prep kits do not require expensive microfluidic equipment or labor-intensive protocols. You can conduct single-cell studies on your benchtop using a simple manual workflow with flexible stopping points.
Simple and scalable workflow
Create templated emulsions using a vortex mixer to capture and barcode single-cell mRNA.
Generate cDNA and prepare single-cell libraries for sequencing.
Sequence on an NGS system to match the scale of your study.
Analyze and visualize single-cell data using DRAGEN and Illumina Connected Multiomics.
Neuroscience
Neuronal tissues comprise a complex organization of diverse cell types that continuously reorganize and remodel throughout an organism’s lifetime. Illumina Single Cell 3' RNA Prep can help researchers catalog the true diversity of neuronal cell populations with transcriptional sensitivity, providing insight into:
Neurodevelopmental and neurodegenerative diseases
Previously unknown roles of specific neuronal populations
The effect of neuro-immune cells on disease and development
Cancer research
Cancer is a dynamic and diverse disease involving complex cell populations. Understanding the cell types and mutations that drive cancer requires sophisticated methods. The Illumina Single Cell 3' RNA Prep kits can help scientists:
Detail heterogeneity of the tumor microenvironment
Identify novel cancer biomarkers
Understand mechanisms of immunotherapy and drug resistance
Immunology
The immune system is composed of a complex hierarchy of diverse cell types that work in concert to identify, target, and eliminate pathogens. Illumina Single Cell 3' RNA Prep kits can help researchers:
Understand developmental and differentiation pathways in immune cell populations
Evaluate the roles and functions of immune cell populations
Study the immune response to pathogens
Example workflow
Illumina Single Cell 3' RNA Prep
NextSeq 550 System
NextSeq 1000 System
NextSeq 2000 System
NovaSeq 6000 System
NovaSeq X Series
DRAGEN Single Cell
DRAGEN Server
Illumina Connected Analytics
Illumina Connected Multiomics
Project recommendations
Instrument: Recommended number of samples
NextSeq 2000 System: Samples per run: P1: 2 T2 samples P2: 10 T2 samples or 2 T10 sample P3: 30 T2 samples, 6 T10 samples, or 3 T20 samples P4: 45 T2 samples, 9 T10 samples, or 4 T20 samples
Samples per run: P1: 2 T2 samples P2: 10 T2 samples or 2 T10 sample P3: 30 T2 samples, 6 T10 samples, or 3 T20 samples P4: 45 T2 samples, 9 T10 samples, or 4 T20 samples
100 cycle kits to run 45 bp × 72 bp
NovaSeq 6000 System: Samples per flow cell (single flowcell run): SP: 20 T2 samples, 4 T10 samples, or 2 T20 samples S1: 40 T2 samples, 8 T10 samples, or 4 T20 samples S2: 102 T2 samples, 20 T10 samples, 10 T20 samples, or 2 T100 samples S4: 250 T2 samples, 50 T10 samples, 25 T20 samples, or 5 T100 samples
Samples per flow cell (single flowcell run): SP: 20 T2 samples, 4 T10 samples, or 2 T20 samples S1: 40 T2 samples, 8 T10 samples, or 4 T20 samples S2: 102 T2 samples, 20 T10 samples, 10 T20 samples, or 2 T100 samples S4: 250 T2 samples, 50 T10 samples, 25 T20 samples, or 5 T100 samples
NovaSeq X Plus System: Samples per flow cell (single flowcell run): 1.5B: 40 T2 samples, 8 T10 samples, or 4 T20 samples 10B: 250 T2 samples, 50 T10 samples, 25 T20 samples, or 5 T100 samples 25B: 650 T2 samples, 130 T10 samples, 65 T20 samples, or 13 T100 samples
Samples per flow cell (single flowcell run): 1.5B: 40 T2 samples, 8 T10 samples, or 4 T20 samples 10B: 250 T2 samples, 50 T10 samples, 25 T20 samples, or 5 T100 samples 25B: 650 T2 samples, 130 T10 samples, 65 T20 samples, or 13 T100 samples
Related applications and methods
Single-cell RNA sequencing
With single-cell RNA-Seq, or scRNA-Seq, you can study cellular differences often masked by bulk sampling. Explore high- and low-throughput single-cell sequencing methods.
Cancer single-cell analysis
Single-cell sequencing allows researchers to understand drivers of cancer at the DNA, RNA, epigenetic, and protein levels within a single cell.
Multiomics
Use multiomics profiling to multiply discovery power and better connect genotype to phenotype.
Figures
Single-cell mRNA capture and barcoding with PIPseq chemistry
Create templated emulsions using a vortex mixer to capture and barcode single-cell mRNA. Generate cDNA and prepare single-cell libraries for sequencing. Sequence on an NGS system to match the scale of your study. Analyze scRNA-Seq data using DRAGEN and Partek software.
High-resolution scRNA-Seq at a scale of thousands of cells
Barcode rank plot (left) and barnyard scatter plot (right) showing effective separation of mixed culture of NIH3T3 (mouse) and HEK 293 (human) cell lines. 4106 cells captured using the Illumina Single Cell 3ʹ RNA Prep T2 kit with less than 5% multiplets.
High-resolution scRNA-Seq at a scale of tens of thousands of cells
scRNA-Seq for cryopreserved human peripheral blood mononuclear cells (PBMCs). UMAP of 31,613 cells captured using the Illumina Single Cell 3ʹ RNA Prep T20 kit resolves diverse immune cell populations.
High-resolution scRNA-Seq at a scale of hundreds of thousands of cells
scRNA-Seq for mouse brain nuclei from frozen tissue. UMAP analysis of 155,000 nuclei captured using the Illumina Single Cell 3ʹ RNA Prep T100 kit resolves diverse brain cell populations.
Resources
The Illumina 3' Single Cell RNA Prep Workflow
Discover how the Illumina 3' Single Cell RNA Prep workflow makes single-cell sequencing simple, affordable, and microfluidics-free. With proven performance across diverse cell types and reliable, high-quality data, it’s easier than ever to decode at single-cell resolution and power your next breakthrough.
Illumina Single Cell 3' RNA Prep, T2 (8 samples, 2000 cells/sample)
20135689
Illumina Single Cell 3' RNA Prep, T2 (8 samples), includes Illumina Single Cell Library Prep plus reagents for single-cell mRNA capture, barcoding, and cDNA synthesis of up to 2000 cells per sample. View less
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Illumina Single Cell 3' RNA Prep, T10 (8 samples, 10,000 cells/sample)
20135691
Illumina Single Cell 3' RNA Prep, T10 (8 samples), includes Illumina Single Cell Library Prep plus reagents for single-cell mRNA capture, barcoding, and cDNA synthesis of up to 10,000 cells per sample. View less
Illumina Single Cell 3' RNA Prep, T20 (4 samples, 20,000 cells/sample)
20135692
Illumina Single Cell 3' RNA Prep, T20 (4 samples), includes Illumina Single Cell Library Prep plus reagents for single-cell mRNA capture, barcoding, and cDNA synthesis of up to 20,000 cells per sample. View less
Illumina Single Cell 3' RNA Prep, T100 (2 samples, 100,000 cells/sample)
20135693
Illumina Single Cell 3' RNA Prep, T100 (2 samples), includes Illumina Single Cell Library Prep plus reagents for single-cell mRNA capture, barcoding, and cDNA synthesis of up to 100,000 cells per sample. View less
Illumina Single Cell Unique Dual Indexes (96 indexes, 96 samples)
20132788
Illumina Single Cell Unique Dual Indexes (96 indexes, 96 samples), optional accessory for use with the Illumina Single Cell Library Prep for high-plex pooling of libraries. View less
Illumina Single Cell Nuclei Isolation Kit (4 samples)
20132795
Illumina Nuclei Isolation Kit (4 samples), optional reagents for nuclei isolation, washing, and resuspension for input into Illumina Single Cell 3' RNA Prep. View less
Illumina Single Cell Supplemental Enrichment and Amplification Kit
20132794
Illumina Single Cell Supplemental Enrichment and Amplification (SEA) Kit, optional reagents for use with Illumina Single Cell 3' RNA Capture and Illumina Single Cell Library Prep for custom single-cell applications. View less
Illumina Single Cell Prep Starter Equipment
20132796
Illumina Single Cell Prep Starter Equipment, one-time purchase required to prepare Illumina Single Cell Library Prep. Includes the Dry Bath device with heated lid and heat blocks for 0.5ml, 1.5ml, 5ml tubes and Vortex mixer device and accessory tube holder assemblies and tube stands for 0.5ml, 1.5ml, 5ml tubes. View less
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How does Illumina Single Cell 3' RNA Prep work?
Illumina Single Cell 3' RNA Prep kits use PIPseq chemistry with particle-templated instant partitions (PIPs). PIPs can segregate complex cell mixtures into partitions with barcoded template particles that can be easily processed for single-cell applications such as single-cell RNA sequencing (scRNA-Seq).
Is the Single Cell Prep Starter Equipment required, and how do the vortex mixer and dry bath differ from what we have?
Yes, our vortex mixer and dry bath are required for reliable results. The vortex mixer is engineered for consistent emulsification. The dry bath ensures precise temperature profiles; typical dry baths do not allow the quick temperature changes that are described in our lysis protocol.
Can fixed cells be used in the Illumina Single Cell 3' RNA Prep workflow?
Yes, using the Fluent DSP/Methanol Fixation demonstrated protocol for cells or nuclei. Paraformaldehyde (PFA) fixation is not compatible currently.
Is 5' mRNA capture available with Illumina Single Cell RNA Prep?
No, only 3' mRNA capture is available currently.
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