CEL-Seq
Cell Expression by Linear Amplification Sequencing
CEL-Seq uses barcoding and pooling of RNA to overcome challenges from low input (Hashimshony et al., 2012). In this method, each cell undergoes RT with a unique barcoded primer in its individual tube. After second-strand synthesis, cDNAs from all reaction tubes are pooled and PCR-amplified. Paired-end deep sequencing of the PCR products allows for accurate detection of sequence information derived from both strands.
Similar methods: CEL-Seq2, Quartz-Seq, Drop-seq, MARS-Seq, CytoSeq, inDrop, Hi-SCL
Advantages:
- Barcoding and pooling allow for multiplexing and studying many different single cells at a time
- Cross-contamination is greatly reduced due to using 1 tube per cell
- Fewer steps than single-cell tagged reverse-transcription sequencing (STRT-Seq)
- Very little read-length bias (Bhargava et al., 2014)
- Strand-specific
Disadvantages:
- Strongly 3′ biased (Shapiro et al., 2013)
- Abundant transcripts are amplified preferentially
- Requires at least 400 pg of total RNA
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
Zhang X., Marjani S. L., Hu Z., Weissman S. M., Pan X., et al. Single-Cell Sequencing for Precise Cancer Research: Progress and Prospects. Cancer Res. 2016;76:1305-1312
Grun D. and van Oudenaarden A. Design and Analysis of Single-Cell Sequencing Experiments. Cell. 2015;163:799-810
Kolodziejczyk A. A., Kim J. K., Svensson V., Marioni J. C. and Teichmann S. A. The Technology and Biology of Single-Cell RNA Sequencing. Mol Cell. 2015;58:610-620
Liang J., Cai W. and Sun Z. Single-Cell Sequencing Technologies: Current and Future. J Genet Genomics. 2014;41:513-528
References:
Levin M., Anavy L., Cole A. G., et al. The mid-developmental transition and the evolution of animal body plans. Nature. 2016;
Bose S., Wan Z., Carr A., Rizvi A. H., Vieira G., et al. (2015) Scalable microfluidics for single-cell RNA printing and sequencing. Genome Biol 16: 120
Seillet C., Mielke L. A., Amann-Zalcenstein D. B., et al. Deciphering the Innate Lymphoid Cell Transcriptional Program. Cell Rep. 2016;17:436-447
Mooijman D., Dey S. S., Boisset J. C., Crosetto N. and van Oudenaarden A. Single-cell 5hmC sequencing reveals chromosome-wide cell-to-cell variability and enables lineage reconstruction. Nat Biotechnol. 2016;
Thomsen E. R., Mich J. K., Yao Z., et al. Fixed single-cell transcriptomic characterization of human radial glial diversity. Nat Methods. 2016;13:87-93
Grun D., Lyubimova A., Kester L., et al. Single-cell messenger RNA sequencing reveals rare intestinal cell types. Nature. 2015;
Klein A. M., Mazutis L., Akartuna I., et al. Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells. Cell. 2015;161:1187-1201
Grun D., Kester L. and van Oudenaarden A. Validation of noise models for single-cell transcriptomics. Nat Methods. 2014;11:637-640
Bhargava V., Head S. R., Ordoukhanian P., Mercola M. and Subramaniam S. Technical variations in low-input RNA-seq methodologies. Sci Rep. 2014;4:3678
Hashimshony T., Feder M., Levin M., Hall B. K. and Yanai I. Spatiotemporal transcriptomics reveals the evolutionary history of the endoderm germ layer. Nature. 2014;
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History: CEL-Seq
Revision by sbrumpton on 2017-06-21 07:50:21 - Show/Hide
Cell Expression by Linear Amplification Sequencing
CEL-Seq uses barcoding and pooling of RNA to overcome challenges from low input (Hashimshony et al., 2012). In this method, each cell undergoes RT with a unique barcoded primer in its individual tube. After second-strand synthesis, cDNAs from all reaction tubes are pooled and PCR-amplified. Paired-end deep sequencing of the PCR products allows for accurate detection of sequence information derived from both strands.
Similar methods: CEL-Seq2, Quartz-Seq, Drop-seq, MARS-Seq, CytoSeq, inDrop, Hi-SCL
Advantages:- Barcoding and pooling allow for multiplexing and studying many different single cells at a time
- Cross-contamination is greatly reduced due to using 1 tube per cell
- Fewer steps than single-cell tagged reverse-transcription sequencing (STRT-Seq)
- Very little read-length bias (Bhargava et al., 2014)
- Strand-specific
Disadvantages:- Strongly 3' biased (Shapiro et al., 2013)
- Abundant transcripts are amplified preferentially
- Requires at least 400 pg of total RNA
Reagents:Illumina Library prep and Array Kit SelectorReviews:Zhang X., Marjani S. L., Hu Z., Weissman S. M., Pan X., et al. Single-Cell Sequencing for Precise Cancer Research: Progress and Prospects. Cancer Res. 2016;76:1305-1312Grun D. and van Oudenaarden A. Design and Analysis of Single-Cell Sequencing Experiments. Cell. 2015;163:799-810Kolodziejczyk A. A., Kim J. K., Svensson V., Marioni J. C. and Teichmann S. A. The Technology and Biology of Single-Cell RNA Sequencing. Mol Cell. 2015;58:610-620Liang J., Cai W. and Sun Z. Single-Cell Sequencing Technologies: Current and Future. J Genet Genomics. 2014;41:513-528References:Levin M., Anavy L., Cole A. G., et al. The mid-developmental transition and the evolution of animal body plans. Nature. 2016;Bose S., Wan Z., Carr A., Rizvi A. H., Vieira G., et al. (2015) Scalable microfluidics for single-cell RNA printing and sequencing. Genome Biol 16: 120Seillet C., Mielke L. A., Amann-Zalcenstein D. B., et al. Deciphering the Innate Lymphoid Cell Transcriptional Program. Cell Rep. 2016;17:436-447Mooijman D., Dey S. S., Boisset J. C., Crosetto N. and van Oudenaarden A. Single-cell 5hmC sequencing reveals chromosome-wide cell-to-cell variability and enables lineage reconstruction. Nat Biotechnol. 2016;Thomsen E. R., Mich J. K., Yao Z., et al. Fixed single-cell transcriptomic characterization of human radial glial diversity. Nat Methods. 2016;13:87-93Grun D., Lyubimova A., Kester L., et al. Single-cell messenger RNA sequencing reveals rare intestinal cell types. Nature. 2015;Klein A. M., Mazutis L., Akartuna I., et al. Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells. Cell. 2015;161:1187-1201Grun D., Kester L. and van Oudenaarden A. Validation of noise models for single-cell transcriptomics. Nat Methods. 2014;11:637-640Bhargava V., Head S. R., Ordoukhanian P., Mercola M. and Subramaniam S. Technical variations in low-input RNA-seq methodologies. Sci Rep. 2014;4:3678Hashimshony T., Feder M., Levin M., Hall B. K. and Yanai I. Spatiotemporal transcriptomics reveals the evolutionary history of the endoderm germ layer. Nature. 2014;