RNA-Seq analysis to capture the transcriptome landscape of a single cell

Author: Tang, F.; Barbacioru, C.; Nordman, E.; Li, B.; Xu, N.; Bashkirov, V.I.; Lao, K.; Surani, M.A.

Description: We describe here a protocol for digital transcriptome analysis in a single mouse oocyte and blastomere using a deep-sequencing approach. In this method, individual cells are isolated and transferred into lysate buffer by mouth pipette, followed by reverse transcription carried out directly on the whole cell lysate. Free primers are removed by exonuclease I and a poly(A) tail is added to the 3′ end of the first-strand cDNAs by terminal deoxynucleotidyl transferase. Single-cell cDNAs are then amplified by 20 + 9 cycles of PCR. The resulting 100-200 ng of amplified cDNAs are used to construct a sequencing library, which can be used for deep sequencing using the SOLiD system. Compared with cDNA microarray techniques, our assay can capture up to 75% more genes expressed in early embryos. This protocol can generate deep-sequencing libraries for 16 single-cell samples within 6 d.

Subject Headings: Animals; Base Sequence; Blastomeres/metabolism; DNA, Complementary/genetics; Female; Gene Expression Profiling/methods; Gene Library; Mice; Molecular Sequence Data; Oligonucleotide Array Sequence Analysis/methods; Oocytes/metabolism; RNA/genetics/isolation & purification; Sequence Analysis, RNA/methods; Nuclease

Publication year: 2010

Journal or book title: Nature Protocols

Volume: 5

Issue: 3

Pages: 516-535

Find the full text : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3847604/

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Type: Journal Article

Serial number: 2829