Transcriptional profiling of day 12 porcine embryonic disc and trophectoderm samples using ultra-deep sequencing technologies.
cDNA derived from trophectoderm (TE) and embryonic disc (ED) of a single day 12 porcine embryo was subjected to next-generation sequencing using the Illumina platform. The short sequencing reads from triplicate sequencing runs were aligned to a custom database designed to represent the known porcine transcriptome. As expected, genes involved in epithelial cell function and steroid biosynthesis were more abundant in cells from the TE; genes involved in maintenance of pluripotency and chromatin remodeling were more highly expressed in cells from the ED. Quantitative real-time PCR was used to confirm the validity of the approach. We conclude that gene expression profiles of even extremely small samples (<or=1,000 cells) can be adequately described without RNA/cDNA preamplification. We also demonstrate the utility of pre-genome genomics resources-such as EST repositories-in the analysis and application of next-generation sequencing data in the absence of an appropriately annotated reference genome.
Isom
S Clay
SC
Division of Animal Science, University of Missouri, Columbia, Missouri, USA. clay.isom@usu.edu
Spollen
William G
WG
Blake
Sean M
SM
Bauer
Bethany K
BK
Springer
Gordon K
GK
Prather
Randall S
RS
D000818:Animals; D001483:Base Sequence; D042002:Chromatin Assembly and Disassembly / Q000235:genetics; D004475:Ectoderm / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D020224:Expressed Sequence Tags; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D008297:Male; D017422:Sequence Analysis, DNA / Q000379:methods*; D013552:Swine / Q000196:embryology* / Q000235:genetics*; D014158:Transcription, Genetic