Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Illumina sequencing of small RNAs from C. elegans embryos

ABSTRACT: Caenorhabditis elegans is one of the most prominent model systems to study embryogenesis. However, it has been impractical to collect large amounts of precisely staged embryos. Thus, early C. elegans embryogenesis has not been amenable to most modern high-throughput genomics or biochemistry assays. To overcome this problem, we devised a method to collect large amounts of cleanly staged C. elegans embryos by Fluorescent Activated Cell Sorting (termed eFACS). eFACS can in principle be applied to all embryonic developmental stages up to hatching. As a proof of principle we show that a single eFACS run routinely yields tens of thousands of almost perfectly staged one-cell embryos. Since in animals the earliest embryonic events are driven by post-transcriptional regulation, we combined eFACS with next-generation sequencing technology to systematically profile the embryonic expression of small, non-coding RNAs. We discovered a wealth of complex and orchestrated changes in the expression between and within almost all classes of small RNAs, including miRNAs, during embryogenesis. Our data indicate that half of all known miRNAs are already expressed in the one-cell stage embryo and we also shed light on the expression and genomic organization of the previously under-appreciated 26G-RNAs. Together, our eFACS data suggest that the complexity of small RNA expression dynamics in animals is comparable to the expression dynamics of protein encoding genes. Various C. elegans embryo samples were generated: mixed embryos by traditional bleaching (Brenner, 1974), early embryos by eFACS (Stoeckius et al., in press). RNA was extracted and length fractionated. Small RNA was subjected to a 5'-dependent ligation protocol to add sequencing adapters. The small RNA samples were sequenced using the Illumina GA I & II.

ORGANISM(S): Caenorhabditis elegans

SUBMITTER: Jonas Maaskola

PROVIDER: E-GEOD-17153 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Large-scale sorting of C. elegans embryos reveals the dynamics of small RNA expression.

Stoeckius Marlon M Maaskola Jonas J Colombo Teresa T Rahn Hans-Peter HP Friedländer Marc R MR Li Na N Chen Wei W Piano Fabio F Rajewsky Nikolaus N

Nature methods 20090906 10

Caenorhabditis elegans is one of the most prominent model systems for embryogenesis, but collecting many precisely staged embryos has been impractical. Thus, early C. elegans embryogenesis has not been amenable to most high-throughput genomics or biochemistry assays. To overcome this problem, we devised a method to collect staged C. elegans embryos by fluorescence-activated cell sorting (eFACS). In a proof-of-principle experiment, we found that a single eFACS run routinely yielded tens of thousa ...[more]

PMID: 19734907

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Project description:In Caenorhabditis elegans, the dosage compensation complex (DCC) specifically binds to and represses transcription from both X chromosomes in hermaphrodites. The DCC is composed of an X-specific condensin complex that interacts with several proteins. During embryogenesis, DCC starts localizing to the X chromosomes around the 40-cell stage, and is followed by X-enrichment of H4K20me1 between 100-cell to comma stage. Here, we analyzed dosage compensation of the X chromosome between sexes, and the roles of dpy-27 (condensin subunit), dpy-21 (non-condensin DCC member), set-1 (H4K20 monomethylase) and set-4 (H4K20 di-/tri-methylase) in X chromosome repression using mRNA-seq and ChIP-seq analyses across several developmental time points. We found that the DCC starts repressing the X chromosomes by the 40-cell stage, but X-linked transcript levels remain significantly higher in hermaphrodites compared to males through the comma stage of embryogenesis. Dpy-27 and dpy-21 are required for X chromosome repression throughout development, but particularly in early embryos dpy-27 and dpy-21 mutations produced distinct expression changes, suggesting a DCC independent role for dpy-21. We previously hypothesized that the DCC increases H4K20me1 by reducing set-4 activity on the X chromosomes. Accordingly, in the set-4 mutant, H4K20me1 increased more from the autosomes compared to the X, equalizing H4K20me1 level between X and autosomes. H4K20me1 increase on the autosomes led to a slight repression, resulting in a relative effect of X derepression. H4K20me1 depletion in the set-1 mutant showed greater X derepression compared to equalization of H4K20me1 levels between X and autosomes in the set-4 mutant, indicating that H4K20me1 level is important, but X to autosomal balance of H4K20me1 contributes only slightly to X-repression. Thus H4K20me1 by itself is not a downstream effector of the DCC. In summary, X chromosome dosage compensation starts in early embryos as the DCC localizes to the X, and is strengthened in later embryogenesis by H4K20me1. RNA-Seq profiles of C. elegans wild type hermaphrodite, mixed sex, at 5 time points and dpy-27, set-4, dpy-21, set-1 and RNAi at 2-3 time points with 3-4 replicates each. RNA-Seq profiles of C. elegans. Strains are N2 (wild type), BS553 fog-2(oz40) V, CB428 dpy-21(e428) V, MK4 dpy-27(y56) III, MT14911 set-4 (n4600) II, SS1075 set-1(tm1821)/hT2g[bli-4(e937) let-?(q782) qIs48] (I;III). Set-1 collections made as heterozygotes and dpy-27(y56) homozygotes. Spike in RNA-seq libraries have AF16 wild type C. briggsae L1s added at a 1:10 ratio. Timepoints are early embryos (synchronized collection, <40 cells), comma embryos (synchronized early embryos aged for 4 hours), mixed embryos (mixed stage embryos from unsynchronized population), L1 (synchronized L1 larvae), L3 (synchronized L3 larvae), and YA (synchronized young adults, collected before embryos present in hermaphrodite gonad). Biological replicates for each strain/stage listed separately. ChIP-seq profiles of C. elegans DCC subunit dpy-27, H4K20me1 histone modification and RNA pol II large subunit ama-1 in 3-6 replicates from mixed stage (unsynchronized) embryos and synchronized L3 larvae. Corresponding inputs are labelled with "Input_" plus ChIP name.

Dataset Information

Illumina sequencing of small RNAs from C. elegans embryos

Publications

Large-scale sorting of C. elegans embryos reveals the dynamics of small RNA expression.

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