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;