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PLoS One. 2013 Aug 12;8(8):e71820. doi: 10.1371/journal.pone.0071820. eCollection 2013.

Sequencing mRNA from cryo-sliced Drosophila embryos to determine genome-wide spatial patterns of gene expression.

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  • 1Graduate Program in Biophysics, University of California, Berkeley, California, United States of America. peter.combs@berkeley.edu

Abstract

Complex spatial and temporal patterns of gene expression underlie embryo differentiation, yet methods do not yet exist for the efficient genome-wide determination of spatial expression patterns during development. In situ imaging of transcripts and proteins is the gold-standard, but it is difficult and time consuming to apply to an entire genome, even when highly automated. Sequencing, in contrast, is fast and genome-wide, but is generally applied to homogenized tissues, thereby discarding spatial information. To take advantage of the efficiency and comprehensiveness of sequencing while retaining spatial information, we cryosectioned individual blastoderm stage Drosophila melanogaster embryos along the anterior-posterior axis and developed methods to reliably sequence the mRNA isolated from each 25 µm slice. The spatial patterns of gene expression we infer closely match patterns previously determined by in situ hybridization and microscopy. We applied this method to generate a genome-wide timecourse of spatial gene expression from shortly after fertilization through gastrulation. We identified numerous genes with spatial patterns that have not yet been described in the several ongoing systematic in situ based projects. This simple experiment demonstrates the potential for combining careful anatomical dissection with high-throughput sequencing to obtain spatially resolved gene expression on a genome-wide scale.

PMID:
23951250
PMCID:
PMC3741199
DOI:
10.1371/journal.pone.0071820
[PubMed - indexed for MEDLINE]
Free PMC Article
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