Jul 06, 2022
Single-cell sequencing and analysis
- 1Columbia University Irving Medical Center
Protocol Citation: Daniele Neri, Lori Zeltser 2022. Single-cell sequencing and analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l61drdvqe/v1
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
featureCounts:
- 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
- featureCounts is available under GNU General Public License as part of the Subread (http://subread.sourceforge.net)
SAMtools:
- Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G., Durbin, R., & 1000 Genome Project Data Processing Subgroup (2009). The Sequence Alignment/Map format and SAMtools. Bioinformatics (Oxford, England),25(16), 2078–2079. https://doi.org/10.1093/bioinformatics/btp352
UMI-tools:
- 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
Sequencing
Sequencing
We performed plate-based single-cell sequencing as described in Snyder et al., 2019 (https://doi.org/10.1126/sciimmunol.aav5581)
Pooled 3’-end sequencing libraries were sequenced on an Illumina NextSeq 500/550 platform
Alignment and count matrix generation
Alignment and count matrix generation
The reads were aligned to mouse genome, GRCm38, gencode version M13 (Ensembl 88) using STAR v2.5.3a
The aligned reads were assigned to genes using featureCounts v1.5.3.
bam files were sorted and indexed using Samtools v1.4.1, duplicated reads were removed and a UMI count matrix was generated using umi tools
Analysis
Analysis
We performed analysis of the count matrices using Seurat v2.3.4 in R v3.4.3 (R Core Team, 2013).