Abstract
Single-cell RNA sequencing (scRNA-seq) experiment reveals previously unseen molecular features. The number of sequencing procedures and computational data analysis approaches has been increasing rapidly in recent years. This chapter provides a general idea of the single-cell data analysis and visualization. An introduction and practical guidance for the 10× sequencing data analysis and visualization are presented. Basic data analysis approaches are highlighted, followed by quality control of data, filtering in cell level and gene level, normalization, dimensional reduction, clustering analysis, and marker identification.
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References
Lieth E, Gardner TW, Barber AJ, Antonetti DA, Penn State Retina Research G (2000) Retinal neurodegeneration: early pathology in diabetes. Clin Exp Ophthalmol 28:3–8. https://doi.org/10.1046/j.1442-9071.2000.00222.x
Zeng XX, Ng YK, Ling EA (2000) Neuronal and microglial response in the retina of streptozotocin-induced diabetic rats. Vis Neurosci 17:463–471. https://doi.org/10.1017/s0952523800173122
Saddala MS, Yang X, Tang S, Huang H (2021) Transcriptome-wide analysis reveals core sets of transcriptional regulators of sensome and inflammation genes in retinal microglia. Genomics 113:3058–3071. https://doi.org/10.1016/j.ygeno.2021.07.001
Cho BP, Song DY, Sugama S, Shin DH, Shimizu Y, Kim SS, Kim YS, Joh TH (2006) Pathological dynamics of activated microglia following medial forebrain bundle transection. Glia 53:92–102. https://doi.org/10.1002/glia.20265
Barcia C, Ros CM, Annese V, Gomez A, Ros-Bernal F, Aguado-Llera D, Martinez-Pagan ME, de Pablos V, Fernandez-Villalba E, Herrero MT (2012) IFN-gamma signaling, with the synergistic contribution of TNF-alpha, mediates cell specific microglial and astroglial activation in experimental models of Parkinson’s disease. Cell Death Dis 3:e379. https://doi.org/10.1038/cddis.2012.123
Ardeljan D, Chan CC (2013) Aging is not a disease: distinguishing age-related macular degeneration from aging. Prog Retin Eye Res 37:68–89. https://doi.org/10.1016/j.preteyeres.2013.07.003
Grigsby JG, Cardona SM, Pouw CE, Muniz A, Mendiola AS, Tsin AT, Allen DM, Cardona AE (2014) The role of microglia in diabetic retinopathy. J Ophthalmol 2014:705783. https://doi.org/10.1155/2014/705783
Liao J, Yu Z, Chen Y, Bao M, Zou C, Zhang H, Liu D, Li T, Zhang Q, Li J, Cheng J, Mo Z (2020) Single-cell RNA sequencing of human kidney. Sci Data 7:4. https://doi.org/10.1038/s41597-019-0351-8
Bykov Y, Kim SH, Zamarin D (2020) Preparation of single cells from tumors for single-cell RNA sequencing. Methods Enzymol 632:295–308. https://doi.org/10.1016/bs.mie.2019.05.057
Wolf FA, Angerer P, Theis FJ (2018) SCANPY: large-scale single-cell gene expression data analysis. Genome Biol 19:15. https://doi.org/10.1186/s13059-017-1382-0
McCarthy DJ, Campbell KR, Lun AT, Wills QF (2017) Scater: pre-processing, quality control, normalization and visualization of single-cell RNA-seq data in R. Bioinformatics 33:1179–1186. https://doi.org/10.1093/bioinformatics/btw777
Butler A, Hoffman P, Smibert P, Papalexi E, Satija R (2018) Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol 36:411–420. https://doi.org/10.1038/nbt.4096
O’Koren EG, Yu C, Klingeborn M, Wong AYW, Prigge CL, Mathew R, Kalnitsky J, Msallam RA, Silvin A, Kay JN, Bowes Rickman C, Arshavsky VY, Ginhoux F, Merad M, Saban DR (2019) Microglial function is distinct in different anatomical locations during retinal homeostasis and degeneration. Immunity 50:723–737 e7. https://doi.org/10.1016/j.immuni.2019.02.007
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The authors would like to thank Chen **aoni and Juthika (University of Missouri, Columbia, Missouri, USA) for her language assistance.
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Saddala, M.S., Mundla, S., Patyal, N., Dash, S. (2023). Single-Cell RNA Sequencing (scRNA-Seq) Data Analysis of Retinal Homeostasis and Degeneration of Microglia. In: Liu, GS., Wang, JH. (eds) Diabetic Retinopathy. Methods in Molecular Biology, vol 2678. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3255-0_6
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DOI: https://doi.org/10.1007/978-1-0716-3255-0_6
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