mRNA-Seq whole-transcriptome analysis of a single cell.
Next-generation sequencing technology is a powerful tool for transcriptome analysis. However, under certain conditions, only a small amount of material is available, which requires more sensitive techniques that can preferably be used at the single-cell level. Here we describe a single-cell digital gene expression profiling assay. Using our mRNA-Seq assay with only a single mouse blastomere, we detected the expression of 75% (5,270) more genes than microarray techniques and identified 1,753 previously unknown splice junctions called by at least 5 reads. Moreover, 8-19% of the genes with multiple known transcript isoforms expressed at least two isoforms in the same blastomere or oocyte, which unambiguously demonstrated the complexity of the transcript variants at whole-genome scale in individual cells. Finally, for Dicer1(-/-) and Ago2(-/-) (Eif2c2(-/-)) oocytes, we found that 1,696 and 1,553 genes, respectively, were abnormally upregulated compared to wild-type controls, with 619 genes in common.
Wellcome Trust-Cancer Research UK Gurdon Institute of Cancer and Developmental Biology, University of Cambridge, Cambridge, UK.
Fuchou
F
Tang
Catalin
C
Barbacioru
Yangzhou
Y
Wang
Ellen
E
Nordman
Clarence
C
Lee
Nanlan
N
Xu
Xiaohui
X
Wang
John
J
Bodeau
Brian B
BB
Tuch
Asim
A
Siddiqui
Kaiqin
K
Lao
M Azim
MA
Surani
D000465:Algorithms; D000818:Animals; D060565:Argonaute Proteins; D001757:Blastomeres / Q000166:cytology / Q000378:metabolism*; D019927:Cyclin E / Q000235:genetics; D053487:DEAD-box RNA Helicases / Q000235:genetics; D018076:DNA, Complementary / Q000138:chemical synthesis / Q000235:genetics; D030561:Databases, Nucleic Acid; D004722:Endoribonucleases / Q000235:genetics; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D051741:Kruppel-Like Transcription Factors / Q000235:genetics; D051379:Mice; D008815:Mice, Inbred Strains; D018345:Mice, Knockout; D008822:Mice, Transgenic; D020411:Oligonucleotide Array Sequence Analysis; D009865:Oocytes / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D020033:Protein Isoforms / Q000235:genetics; D011506:Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D043244:Ribonuclease III; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods*; D015854:Up-Regulation / Q000235:genetics
C526735:Ago2 protein, mouse; D060565:Argonaute Proteins; D019927:Cyclin E; D018076:DNA, Complementary; C507362:Dppa5 protein, mouse; D015852:Eukaryotic Initiation Factor-2; C498314:Klf2 protein, mouse; D051741:Kruppel-Like Transcription Factors; D020033:Protein Isoforms; D011506:Proteins; D012333:RNA, Messenger; D004722:Endoribonucleases; C509781:Dicer1 protein, mouse; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases