License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: April 01, 2022
Last Modified: July 06, 2022
Protocol Integer ID: 60200
Abstract
This protocol describes single-cell sequencing and analysis.
Materials
References:
Plate-based single-cell sequencing:
Snyder, M. E., Finlayson, M. O., Connors, T. J., Dogra, P., Senda, T., Bush, E., Carpenter, D., Marboe, C., Benvenuto, L., Shah, L., Robbins, H., Hook, J. L., Sykes, M., D'Ovidio, F., Bacchetta, M., Sonett, J. R., Lederer, D. J., Arcasoy, S., Sims, P. A., & Farber, D. L. (2019). Generation and persistence of human tissue-resident memory T cells in lung transplantation. Science Immunology,4(33), eaav5581. https://doi.org/10.1126/sciimmunol.aav5581
STAR:
Dobin, A., Davis, C. A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M., & Gingeras, T. R. (2013). STAR: ultrafast universal RNA-seq aligner. Bioinformatics (Oxford, England),29(1), 15–21. https://doi.org/10.1093/bioinformatics/bts635
Liao, Y., Smyth, G. K., & Shi, W. (2014). featureCounts: an efficient general-purpose program for assigning sequence reads to genomic features. Bioinformatics (Oxford, England),30(7), 923–930. https://doi.org/10.1093/bioinformatics/btt656
Smith, T., Heger, A., & Sudbery, I. (2017). UMI-tools: modeling sequencing errors in Unique Molecular Identifiers to improve quantification accuracy. Genome Research,27(3), 491–499. https://doi.org/10.1101/gr.209601.116
R toolkit Seurat:
Butler, A., Hoffman, P., Smibert, P., Papalexi, E., & Satija, R. (2018). Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nature Biotechnology,36(5), 411–420. https://doi.org/10.1038/nbt.4096