An essential requirement for single-cell RNA sequencing in cancer is the preparation of high-quality single-cell suspensions from the tumor tissue. In this work, various methods of dissociation of tumor biopsy specimens were analyzed and developed to obtain a cell suspension with at least 80% viability. It was found that the optimal conditions for sample preparation are mechanical dissociation followed by incubation with a collagenase/hyaluronidase mixture with addition of DNAase I for 60 min. Thus, we optimize the approach for preparing single-cell suspensions from the tumor biopsy tissue for single-cell RNA sequencing.
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Chen G, Ning B, Shi T. Single-Cell RNA-Seq Technologies and Related Computational Data Analysis. Front. Genet. 2019;10:317. https://doi.org/10.3389/fgene.2019.00317
Potter SS. Single-cell RNA sequencing for the study of development, physiology and disease. Nat. Rev. Nephrol. 2018;14(8):479-492. https://doi.org/10.1038/s41581-018-0021-7
Ilicic T, Kim JK, Kolodziejczyk AA, Bagger FO, McCarthy DJ, Marioni JC, Teichmann SA. Classification of low quality cells from single-cell RNA-seq data. Genome Biol. 2016;17:29. https://doi.org/10.1186/s13059-016-0888-1
Madden RE, Burk D. Production of viable single cell suspensions from solid tumors. J. Natl. Cancer Inst. 1961;841-861.
Quatromoni JG, Singhal S, Bhojnagarwala P, Hancock WW, Albelda SM, Eruslanov E. An optimized disaggregation method for human lung tumors that preserves the phenotype and function of the immune cells. J. Leukoc. Biol. 2015;97(1):201-209. https://doi.org/10.1189/jlb.5TA0814-373.
Rodriguez de la Fuente L, Law AMK, Gallego-Ortega D, Valdes-Mora F. Tumor dissociation of highly viable cell suspensions for single-cell omic analyses in mouse models of breast cancer. STAR Protoc. 2021;2(4):100841. https://doi.org/10.1016/j.xpro.2021.100841
Matta BM, Reichenbach DK, Blazar BR, Turnquist HR. Alarmins and Their Receptors as Modulators and Indicators of Alloimmune Responses. Am. J. Transplant. 2017;17(2):320-327. https://doi.org/10.1111/ajt.1388
Volovitz I, Shapira N, Ezer H, Gafni A, Lustgarten M, Alter T, Ben-Horin I, Barzilai O, Shahar T, Kanner A, Fried I, Veshchev I, Grossman R, Ram Z. A non-aggressive, highly efficient, enzymatic method for dissociation of human brain-tumors and brain-tissues to viable single-cells. BMC Neurosci. 2016;17(1):30. https://doi.org/10.1186/s12868-016-0262-y
Abaffy P, Lettlova S, Truksa J, Kubista M, Sindelka R. Preparation of single-cell suspension from mouse breast cancer focusing on preservation of original cell state information and cell type composition. bioRxiv. 2019;824714. https://doi.org/10.1101/824714
Haque A, Engel J, Teichmann SA, Lönnberg T. A practical guide to single-cell RNA-sequencing for biomedical research and clinical applications. Genome Med. 2017;9(1):75. https://doi.org/10.1186/s13073-017-0467-4
Petit V, Massonnet G, Maciorowski Z, Touhami J, Thuleau A, Némati F, Laval J, Château-Joubert S, Servely JL, Vallerand D, Fontaine JJ, Taylor N, Battini JL, Sitbon M, Decaudin D. Optimization of tumor xenograft dissociation for the profiling of cell surface markers and nutrient transporters. Lab. Invest. 2013;93(5):611-621. https://doi.org/10.1038/labinvest.2013.44
O’Flanagan CH, Campbell KR, Zhang AW, Kabeer F, Lim JLP, Biele J, Eirew P, Lai D, McPherson A, Kong E, Bates C, Borkowski K, Wiens M, Hewitson B, Hopkins J, Pham J, Ceglia N, Moore R, Mungall AJ, McAlpine JN; CRUK IMAXT Grand Challenge Team; Shah SP, Aparicio S. Dissociation of solid tumor tissues with cold active protease for single-cell RNA-seq minimizes conserved collagenase-associated stress responses. Genome Biol. 2019;20(1):210. https://doi.org/10.1186/s13059-019-1830-0
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Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 175, No. 4, pp. 523-528, April, 2023
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Frolova, A.A., Gerashchenko, T.S., Patysheva, M.R. et al. Preparation of a Single-Cell Suspension from Tumor Biopsy Samples for Single-Cell RNA Sequencing. Bull Exp Biol Med 175, 519–523 (2023). https://doi.org/10.1007/s10517-023-05898-9
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DOI: https://doi.org/10.1007/s10517-023-05898-9