NET-Seq

scRNA-Seq

scRNA-Seq provides deeper insight to the multi-tiered complexity of different cells within the same tissue type. scRNA-Seq has now been adapted widely into other methods in the single-cell RNA sequencing field. In this method, single cells are isolated manually under a microscope and lysed. Next, mRNAs are purified and primed with a poly(T) primer for reverse transcription. Unreactive primers are removed by exonuclease I digestion. Poly(A) tails are added to the first strand cDNA at the 3' end and annealed to poly(T) primers for second-strand cDNA generation. Finally, the cDNAs are PCR-amplified, sheared, and prepared into sequencing libraries.

Pros:

• Single-cell–resolution transcriptomic analysis
• Able to detect unknown splice junctions
• Various RNAPII-specific antibodies can be used to increase targeting accuracy

Cons:

• Very low throughput
• No molecular barcodes used
• Can be expensive to scale up

• mNET-Seq: Nojima T., Gomes T., Grosso A. R., et al. Mammalian NET-Seq Reveals Genome-wide Nascent Transcription Coupled to RNA Processing. Cell. 2015;161:526-540
• 1. Nojima T., Gomes T., Carmo-Fonseca M. and Proudfoot N. J. Mammalian NET-seq analysis defines nascent RNA profiles and associated RNA processing genome-wide. Nat Protoc. 2016;11:413-428

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2. Woodhouse S., Moignard V., Gottgens B. and Fisher J. Processing, visualising and reconstructing network models from single-cell data. Immunol Cell Biol. 2016;94:256-265
3. Wagner A., Regev A. and Yosef N. Revealing the vectors of cellular identity with single-cell genomics. Nat Biotechnol. 2016;34:1145-1160
4. Stegle O., Teichmann S. A. and Marioni J. C. Computational and analytical challenges in single-cell transcriptomics. Nat Rev Genet. 2015;16:133-145
5. 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

1. Lee J. H., Daugharthy E. R., Scheiman J., et al. Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues. Nat Protoc. 2015;10:442-458
2. Padovan-Merhar O., Nair G. P., Biaesch A. G., et al. Single Mammalian Cells Compensate for Differences in Cellular Volume and DNA Copy Number through Independent Global Transcriptional Mechanisms. Mol Cell. 2015;58:339-352