Single-Cell RNA Sequencing and Its Combination with Protein and DNA Analyses
Abstract
:1. Introduction
2. Conventional scRNA-seq Technologies
2.1. Smart-seq 1 and 2
2.2. SCRB-seq
2.3. CEL-seq 1 and 2
2.4. MARS-seq 1 and 2
2.5. Quartz-seq 1 and 2
2.6. SUPeR-seq
2.7. MATQ-seq
3. Microfluidic-Based scRNA-seq Technologies
3.1. Valve-Based scRNA-seq Technologies
3.1.1. Multilayer Microfluidic Device for scRNA-seq
3.1.2. Microfluidic Hydrodynamic Trap Array for scRNA-seq
3.1.3. MID-RNA-seq
3.1.4. Hydro-seq
3.2. Droplet-Based scRNA-seq Technologies
3.2.1. Hi-SCL
3.2.2. In-Drop
3.2.3. Drop-seq
3.2.4. 10x Genomics
3.2.5. MULTI-seq
3.3. Nanowell-Based scRNA-seq Technologies
3.3.1. Cytoseq
3.3.2. Microwell-seq
3.3.3. Seq-Well
3.3.4. SCOPE-seq
3.3.5. scFTD-seq
4. Combination of scRNA-seq with Proteomic Analysis
4.1. Cite-seq
4.2. Reap-seq
4.3. PDMS Nanowell and seq
5. Combination of scRNA-seq with DNA Analysis
5.1. DR-seq
5.2. G and T-seq
5.3. SIDR-seq
5.4. CORTAD-seq
5.5. scTrio-seq
6. Commercial scRNA-seq Technologies
7. Conclusions
Author Contributions
Funding
Conflicts of Interest
References
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Technology | Cell Isolation Method | No. of Cells | Cell Barcode | Unique Molecular Identifiers | cDNA Coverage | Amplification Method | Advantages | Limitations | Outcomes |
---|---|---|---|---|---|---|---|---|---|
Smart-seq 1 & 2 [37,38] | Micropipette | 100–1000 | No | No | Full-length | Template switching-based PCR | Increased throughput and read coverage across transcripts Smart-seq 2 increases thermal stability of LNA-DNA base pairs. | Low number of cells Time-consuming cell isolation processes | Transcript enumeration Analysis of alternative splicing allelic expression Investigation of transcriptomic profile in rare cells |
CEL-seq 1 and 2 [13,14] | Micropipette | 100–1000 | Yes | Yes | 3′ tag | In vitro transcription-based 3′ transcript amplification * The protocol is based on Smart-seq | CEL-Seq 2 adds a 5-base pair UMI upstream of the barcode to distinguish between PCR duplicates and transcript abundance in scRNA-seq, which significantly improves accuracy. | 3′ end sequencing only The use of micropipette for cell isolation makes the operational processes more difficult and time-consuming. Low number of cells are processed. | It is used to study early C. elegans embryonic development at single cell level. CEL-seq will be useful for transcriptomic analyses of complex tissues containing populations of diverse cell types. |
SCRB-seq [15] | FACS | 1000–10,000 | Yes | Yes | 3′ tag | Template switching-based PCR * The protocol is based on Smart-seq. | High throughput | Requires skilled workers | Characterization of primary human adipose-derived stem cell differentiation system Discovery of transcriptomes across heterogeneous populations |
MARS-seq 1 & 2 [39,40] | FACS | 1000–5000 | Yes | Yes | 3′ tag | In vitro transcription-based 3′ transcript amplification | Automated processes minimize amplification bias and labeling errors | Requires skilled workers | Analysis of in vivo transcriptional states in thousands of single cells. Identification of a unique microglia type that may restrict the development of Alzheimer’s disease |
Quartz-seq 1 [41] | FACS | 1000–10,000 | No | No | Full length with 3′ biased | PCR after poly(A) tailing | Highly quantitative | Requires skilled workers | Detection of transcriptome heterogeneity between the cells in the same and different cell-cycle phases |
Quartz-seq 2 [42] | FACS | 1000–10,000 | Yes | Yes | Full length with 3′ biased | PCR after poly(A) tailing | Able to detect more transcripts from limited sequence reads at a minimal cost | Requires skilled workers | Detection of transcriptome heterogeneity between embryonic stem cells and between cells in stromal vascular fraction |
SUPeR-seq [43] | Mouth pipette | ~10 | Yes | No | Full length | PCR after poly(A) tailing | Able to detect both circular RNA (non-polyadenylated RNA) and polyadenylated RNA | Low throughput Operational processes are difficult and time-consuming | Analysis of expression dynamics of circular RNA during mammalian early embryonic development |
MATQ-seq [44] | Mouth pipette | 10–100 | Yes | Yes | Full length | PCR after poly(A) tailing | Able to sequence both polyadenylated and non-polyadenylated RNAs with high sensitivity and accuracy | Low throughput Operational processes are difficult and time-consuming | Detection of low abundance genes and non-polyadenylated RNA extracted from a single cell |
Technology | Cell Isolation Method | No. of Cells | Cell Barcode | Unique Molecular Identifiers | cDNA Coverage | Amplification Method | Advantages | Limitations | Outcomes |
---|---|---|---|---|---|---|---|---|---|
Multilayer microfluidic device and seq [19] | Valve | 10–100 | Yes | No | Full-length | PCR after poly(A) tailing | Improvement of assay sensitivity The semi-automated processes minimize technical variation and reduce risk of contamination | The requirement of off-chip amplification Complex device fabrication processes Low throughput | Identification of differentially expressed genes of single cells and measurement of biological variations in cell populations. |
Microfluidic hydrodynamic trap array & seq [50] | Valve | 10–5000 | Yes | No | Full-length | Template switching-based PCR * The protocol is based on Smart-seq 2 | Allows multi-generational lineage tracking under controlled culture conditions | Complex device fabrication processes | Measurement of the effects of lineage and cell cycle-dependent transcriptional profiles of single cells. |
MID-RNA-seq [51] | Valve | 1000 | Yes | No | Full length | PCR after poly(A) tailing | Allows automated processing and multiplexing | Complex device fabrication processes | Transcriptomic studies of scarce cell samples. |
Hydro-seq [52] | Valve | 10–1000 | Yes | Yes | 3′ tag | Template switching-based PCR * The protocol is based on Drop-seq. | Improved throughput and cell capture efficiency | Complex device fabrication processes | Identification of cellular heterogeneity in critical biomarkers of tumor metastasis, understanding tumor metastasis processes, and monitoring target therapeutics in cancer patients. |
Hi-SCL [53] | Droplet | 1000–10,000 | Yes | No | 3′ tag | PCR after poly(A) tailing | High throughput | Low cell capture efficiency | Detection and comparison of transcriptomes in mouse embryonic stem cells and mouse embryonic fibroblast populations at the single-cell level. |
In-drop [24] | Droplet | 1000–10,000 | Yes | Yes | 3′ tag | In vitro transcription-based 3′ transcript amplification * The protocol is based on CEL-seq. | High throughput | Low cell capture efficiency Only the 3′ most terminal fragments can be used for sequencing | Sequencing of large numbers of cells from heterogeneous populations in a fast way and identification of very rare cell types. |
Drop-seq [23] | Droplet | 1000–10,000 | Yes | Yes | 3′ tag | Template switching-based PCR | High throughput, cheaper, and faster | Only the 3′ most terminal fragments can be used for sequencing | Analysis of mRNA transcripts from thousands of individual cells concurrently while identifying the cell of origin. |
10x Genomics [54] | Droplet | 1000–10,000 | Yes | Yes | 3′ tag | Template switching-based PCR | The use of 10x barcodes significantly increase throughput | Only the 3′ most terminal fragments can be used for sequencing | Profile 68k peripheral blood mononuclear cells and dissect large immune populations. |
MULTI-seq [55] | Droplet | 10,000–100,000 | Barcoded lipid-modified oligonucleotides | Yes | 3′ tag | Template switching-based PCR * The protocol is based on 10x genomics. | Readily multiplex various cell types and identify cell doublets | Only the 3′ most terminal fragments can be used for sequencing | Assessment of immune cell responses to tumor metastatic progression. |
Cytoseq [56] | Nanowell | 100–10,000 | Yes | Yes | 3′ tag | Gene specific primers-based PCR | High throughput Simple fabrication and operation processes | Not fully automated | Characterization of cellular heterogeneity in immune response and identification of rare cells in a cell population. |
Microwell-seq [26] | Nanowell | 100–10,000 | Yes | No | Full-length | Template switching-based PCR * The protocol is based on Smart-seq 2. | High throughput Simple fabrication and operation processes | Not fully automated | Construction of “mouse cell atlas” with more than 400k single-cell transcriptomic profiles from 51 mouse tissues, organs, and cell cultures, covering more than 800 major cell types and 1000 cell subtypes in the mouse system. |
Seq-well [25] | Nanowell | 100–10,000 | Yes | Yes | 3′ tag | Template switching-based PCR * The protocol is based on Drop-seq. | High throughput Simple fabrication and operation processes The use of semipermeable polycarbonate membrane reduces well-to-well contamination | Not fully automated | Profile thousands of primary human macrophages exposed to Mycobacterium tuberculosis. It is compatible with on-array imaging cytometry for resolving the phenotype of cells from complex samples. |
SCOPE-seq [57] | Nanowell | 100–10,000 | Yes | Yes | 3′ tag | Template switching-based PCR * The protocol is based on Drop-seq. | High throughput Simple fabrication and operation processes The phenotypes measured can be directly linked to expression profiles using optically decodable beads The use of perfluorinated oil prevents well-to-well contamination | Not fully automated | Combination of live cell imaging with single-cell RNA sequencing for various biomedical applications. |
scFTD-seq [58] | Nanowell | 100–10,000 | Yes | Yes | 3′ tag | Template switching-based PCR * The protocol is based on drop-seq. | High throughput Simple fabrication and operation processes Minimizing contamination by preventing immediate cell lysis | Not fully automated | Profile circulating follicular helper T cells implicated in systemic lupus erythematosus pathogenesis |
Technology | Cell Isolation Method | No. of Cells | Cell Barcode | Unique Molecular Identifiers | cDNA Coverage | cDNA Amplification Method | Advantages | Limitations | Outcomes |
---|---|---|---|---|---|---|---|---|---|
Cite-seq [59] | Droplet | 1000–10,000 | Yes | No | 3′ tag | Template switching-based PCR *The protocol is based on Drop-seq. | High throughput Allows simultaneous transcriptomic and surface protein analysis | Low cell capture efficiency Not fully automated | Simultaneous detection of about 13 surface proteins and transcripts |
Reap-seq [60] | Droplet | 1000–10,000 | Yes | Yes | 3′ tag | Template switching-based PCR * The protocol is based on 10x genomics. | High throughput Allows simultaneous transcriptomic and surface protein analysis | Low cell capture efficiency Not fully automated | Assessment of costimulatory effects of a CD27 agonist on human CD8+ lymphocytes and characterization of an unknown cell type |
PDMS Nanowells and seq [61] | Nanowell | 1000–10,000 | Yes | No | Full length | Template switching-based PCR * The protocol is based on Smart-seq 2. | High throughput Allows simultaneous transcriptomic and secretion analysis | Not fully automated | Study of regulation mechanisms of the immune system |
Technology | Cell Isolation Method | No. of Cells | Cell Barcode | Unique Molecular Identifiers | cDNA Coverage | cDNA Amplification Method | Advantages | Limitations | Outcomes |
---|---|---|---|---|---|---|---|---|---|
DR-seq [65] | Mouth pipette | 10–50 | Yes | No | 3′ tag | In vitro transcription * The protocol is based on CEL-seq. | Allows simultaneous transcriptomic and DNA analysis | Complex work flow and low throughput Requires in silico masking of coding sequences, which complicates the data analysis processes | Study of transcriptional consequences of gDNA copy number variations in diseased and healthy tissues |
G and T-seq [66] | FACS | 10–100 | No | No | Nearly full length | Template switching-based PCR * The protocol is based on Smart-seq 2. | Simple work flow Allows simultaneous transcriptomic and DNA analysis | Requires skilled workers Low throughput | Study of transcriptional consequences of chromosomal abnormalities in a single cell |
SIDR-seq [67] | Micropipette | 10–100 | No | No | Nearly full-length | Template switching-based PCR * The protocol is based on Smart-seq 2. | Automated and simple work flow Allows simultaneous transcriptomic and DNA analysis | Low throughput | Assessment of cellular heterogeneity in breast and lung cancer at the singular cell level |
CORTAD-seq [64] | Fludigm C1 | 100–1000 | No | No | Full length with weak 3′-biased | Template switching-based PCR * The protocol is based on Smart-seq. | Automated and high throughput Allows simultaneous transcriptomic and DNA analysis | Not suitable for genome-wide DNA analysis for discovery purpose | Study of transcriptional consequences of known targeted gene mutations in various types of cancer |
scTrio-seq [68] | Mouth pipette | 10–50 | No | No | Full length with weak 3′-biased | Template switching-based PCR * The protocol is based on Smart-seq. | Allows simultaneous transcriptomic, genomic, and epigenomic analysis | Complex work flow and low throughput | Study of transcriptional consequences of genomic and epigenomic heterogeneities within a population of cells especially cancer cells |
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Choi, J.R.; Yong, K.W.; Choi, J.Y.; Cowie, A.C. Single-Cell RNA Sequencing and Its Combination with Protein and DNA Analyses. Cells 2020, 9, 1130. https://doi.org/10.3390/cells9051130
Choi JR, Yong KW, Choi JY, Cowie AC. Single-Cell RNA Sequencing and Its Combination with Protein and DNA Analyses. Cells. 2020; 9(5):1130. https://doi.org/10.3390/cells9051130
Chicago/Turabian StyleChoi, Jane Ru, Kar Wey Yong, Jean Yu Choi, and Alistair C. Cowie. 2020. "Single-Cell RNA Sequencing and Its Combination with Protein and DNA Analyses" Cells 9, no. 5: 1130. https://doi.org/10.3390/cells9051130
APA StyleChoi, J. R., Yong, K. W., Choi, J. Y., & Cowie, A. C. (2020). Single-Cell RNA Sequencing and Its Combination with Protein and DNA Analyses. Cells, 9(5), 1130. https://doi.org/10.3390/cells9051130