Single cell RNA sequencing (ScRNA-seq) is a powerful tool developed to identify gene expression of individual cells in complex biological tissues. Conventional assays assume homogeneity, averaging gene characteristics across all cells within a sample. This bulk approach fails to capture information essential to understanding the structure and function of the tissue. With the introduction of high-throughput single cell isolation and analysis, researchers have identified rare cell types and compiled gene expression data driving new therapeutics.
Harrick Plasma cleaners are reported in several of the most commonly employed single cell sequencing platforms, including Drop-Seq, Seq-Well and Drop-well. Plasma cleaners are used in the fabrication of microfluidic devices and are essential for rapid prototyping. Because single cell sequencing is a relatively new field, iterative changes to these platforms are needed to improve efficiency, sensitivity and throughput. Researchers may also need to adjust channel size and structure of established platforms to optimize cell sorting and analysis for their target cell type.
The Drop-Seq ScRNA methodology was developed by the McCarroll Lab at Harvard University in 2015 and has become an open-source, keystone methodology in single cell sequencing. This method uses a molecular barcoding strategy to identify individual cells. Barcode beads and single cells are combined in a co-flow microfluidic devices, creating a nanoliter sized reaction chamber for further analysis. As a result, drop-seq can be used to analyze mRNA transcripts from thousands of individual cells simultaneously and can recall the transcripts original cell.
The McCarroll Lab (Open Source) is an excellent resource for researchers planning to employee the Drop-seq platform in their own lab. In addition, below is a sample of papers that report using Harrick Plasma Cleaner’s to fabricate their drop-seq microfluidic devices.
Dirkse A, Golebiewska A, Buder T, et al. Stem cell-associated heterogeneity in Glioblastoma results from intrinsic tumor plasticity shaped by the microenvironment. Nat Commun. 2019;10(1):1787. 10.1038/s41467-019-09853-z
Golebiewska A, Hau A C, Oudin A, Stieber D, Yabo Y, ET AL. “Patient-derived organoids and orthotopic xenografts of primary and recurrent gliomas represent relevant patient avatars for precision oncology”. Acta Neuropathologica 2020 140: 919-949 10.1007%2Fs00401-020-02226-7
Li L, Wu P, Luo Z, Wang L, Ding W, Wu T, Chen J, He J, He Y, Wang H, Chen Y, Li G, Li Z, and He L. “Dean flow assisted single cell and bead encapsulation for high performance single cell expression profiling”. ACS Sensors 2019 4: 1299-1305 10.1021/acssensors.9b00171
Macosko EZ, Basu A, Satija R, et al. “Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets”. Cell 5:1202-1214. 10.1016/j.cell.2015.05.002
Sousa C, Golebiewska A, Poovathingal SK, Kaoma T, Pires-Afonso Y, Martina S, Coowar D, Azuaje F, Skupin A, Balling R, Biber K, Niclou SP, and Michelucci A. “Single-cell transcriptomics reveals distinct inflammation-induced microglia signatures”. EMBO Rep. 2018 19: e46171 10.15252/embr.201846171
Walter J, Bolognin S, Antony PMA, et al. “Neural Stem Cells of Parkinson’s Disease Patients Exhibit Aberrant Mitochondrial Morphology and Functionality”. Stem Cell Reports 2019 12(5):878-889. 10.1016/j.stemcr.2019.03.004
The Seq-Well ScRNA platform was developed by the Shalek lab at MIT and is being developed to analyze low input clinical samples. In SeqWell, mRNA capture beads are loaded into a microwell device to rapidly profile single cell transcriptomes in parallel.
The Shalek Lab website provides extensive tutorial videos for the Seq-Well methodology, enabling researchers to employ the technology in their own labs. Additionally, below is a list of papers using Harrick Plasma Cleaners in Seq-Well.
Gierahn TM, Wadsworth II MH, Hughes TK, Bryson BD, Butler A, Satija R, Fortune S, Love JC, and Shalek AK. “Seq-Well: portable, low-cost RNA sequencing of single cells at high throughput”. Nat. Methods 2017 14: 395 10.1038/nmeth.4179
Ma S, de la Fuente Revenga M, Sun Z, Sun C, Murphy TW, Xie H, González-Maeso J, and Lu C. “Cell-type-specific brain methylomes profiled via ultralow-input microfluidics”. Nat. Biomed. Eng. 2018 2: 183-194 10.1038/s41551-018-0204-3
Numerous single cell sequencing platforms are currently in-development, either created from scratch or through improvement or combination of the Drop-Well or Seq-Well platforms. Below are featured articles with innovative new single cell sequencing platforms.
Other Platform Articles
Delley C L, and Abate A R. “Microfluidic particle zipper enables controlled loading of droplets with distinct particle types”. Lab on a Chip 2020 20: 2465-2472 10.1039/D0LC00339E
Isozaki A, Nakagawa Y, Loo M. H, Shibata Y, Tanaka N, Setyaningrum D L, Park J W, Shirasaki Y, Mikami H, Huang D, Tsoi H, et al. “Sequentially addressable dielectrophoretic array for high-throughput sorting of large-volume biological compartments”. Science Advances 2020 116 10.1126/sciadv.aba6712
Sarma M, Lee J, Ma S, Li S, and Lu C. “A diffusion-based microfluidic device for single-cell RNA-seq”. Lab on a Chip 2019 19:1247-1256 10.1039/C8LC00967H
Samlali K, Ahmadi F, Quach A B V, Soffer G, and Shih S C C. “One Cell, One Drop, One Click: Hybrid Microfluidics for Mammalian Single Cell Isolation”. Small 2020 16: 2002400. 10.1002/smll.202002400