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SRP136035
Caenorhabditis elegans
148 Downloadable Samples
NextSeq 500
Description
Bleaching gravid C. elegans followed by a short period of starvation of the L1 larvae is a routine method performed by worm researchers for generating synchronous populations for experiments. During the process of investigating dietary effects on gene regulation in L1 stage worms by single-worm RNA-Seq, we found that the density of resuspended L1 larvae affects expression of many mRNAs. Specifically, a number of genes related to metabolism and signalling are highly expressed in worms arrested at low density, but are repressed at higher arrest densities. We generated a GFP reporter strain based on one of the most density-dependent genes in our dataset – lips-15 – and confirmed that this reporter was expressed specifically in worms arrested at relatively low density. Finally, we show that conditioned media from high density L1 cultures was able to downregulate lips-15 even in L1 animals arrested at low density, and experiments using daf-22 mutant animals demonstrated that this effect is not mediated by the ascaroside family of signalling pheromones. Together, our data implicate a soluble signalling molecule in density sensing by L1 stage C. elegans, and provide guidance for design of experiments focused on early developmental gene regulation. Overall design: L1 Larvae arrested in M9 media at different densities were isolated for single L1 RNA-sequencing
Publication Title
Effects of Larval Density on Gene Regulation in <i>Caenorhabditis elegans</i> During Routine L1 Synchronization.
Sample Metadata Fields
Cell line, Subject
View Samples- Caenorhabditis elegans
- 10 Downloadable Samples
- Illumina HiSeq 2000, Illumina Genome Analyzer IIx
Description
During each life cycle germ cells preserve and pass on both genetic and epigenetic information. In C. elegans, the ALG-3/4 Argonaute (AGO) proteins and their small-RNA cofactors are expressed during male gametogenesis and promote male fertility. Here we show that the CSR-1 AGO functions with ALG-3/4 to positively regulate target genes required for spermiogenesis. Our findings suggest that ALG-3/4 functions during spermatogenesis to amplify a small-RNA signal that represents an epigenetic memory of male-specific gene expression, while CSR-1, which is abundant in mature sperm, transmits this memory to offspring. Surprisingly, in addition to small RNAs targeting male-specific genes, we show that males also harbor an extensive repertoire of CSR-1 small RNAs targeting oogenesis-specific mRNAs. Together these findings suggest that C. elegans sperm transmit not only the genome but also epigenetic binary signals in the form of Argonaute/small-RNA complexes that constitute a memory of which genes were active in preceding generations. Overall design: Examine small RNA changes in WT and alg-3/4 mutant males cultured at 20°C and 25°C, as well as determine the small RNAs enriched in a FLAG::CSR-1 IP from male worms grown at 25°C. mRNA sequencing was also performed to determine how transcripts targeted by small RNAs change in mutant background at 20°C and 25°C.
Publication Title
Argonautes promote male fertility and provide a paternal memory of germline gene expression in C. elegans.
Sample Metadata Fields
Subject
View Samples- Mus musculus
- 932 Downloadable Samples
- NextSeq 500
Description
The small RNA payload of mammalian sperm undergoes dramatic remodeling during development, as several waves of microRNAs and tRNA fragments are shipped to sperm during post-testicular maturation in the epididymis. Here, we take advantage of this developmental process to probe the function of the sperm RNA payload in preimplantation development. We generated zygotes via intracytoplasmic sperm injection (ICSI) using sperm obtained from the proximal (caput) vs. distal (cauda) epididymis, then characterized development of the resulting embryos. Embryos generated using caput sperm significantly overexpress multiple regulatory factors throughout preimplantation development, and subsequently implant inefficiently and fail soon after implantation. Remarkably, microinjection of purified cauda-specific small RNAs into caput-derived embryos not only completely rescued preimplantation molecular defects, but also suppressed the postimplantation embryonic lethality phenotype. These findings reveal an essential role for small RNA remodeling during post-testicular maturation of mammalian sperm, and identify a specific preimplantation gene expression program responsive to sperm-delivered microRNAs. Overall design: Zygotes were generated by ICSI from sperm isolated from the testis, caput epididymis, or cauda epididiymis. Following fertilization by ICSI zygotes were developed to different stages of preimplantation development and were harvested for single-embryo RNA-Seq (Smart-Seq 2 protocol). For RNA injection experiments 3 hours after fertilization by ICSI RNA was injected using an Eppendorf Femtojet injection setup.
Publication Title
Small RNAs Gained during Epididymal Transit of Sperm Are Essential for Embryonic Development in Mice.
Sample Metadata Fields
Cell line, Subject
View Samples- Caenorhabditis elegans
- 4 Downloadable Samples
- Illumina Genome Analyzer II
Description
High-throughput pyrosequencing of endogenous small RNAs from >95% male enriched populations of alg-3(tm1155);alg-4(ok1041);fog-2(q71) and fog-2(q71) worms as well as purified spermatids from fem-3(q20) adult worms. Gametogenesis is thermosensitive in numerous metazoa ranging from worms to man. In C. elegans a variety of germ-line nuage- (P-granule) -associated RNA-binding proteins including the Piwi-clade Argonaute, PRG-1, have been implicated in temperature-dependent fertility. Here, we describe the role of two AGO-class paralogs, alg-3 (T22B3.2) and alg-4 (ZK757.3) in promoting male fertility at elevated temperatures. A rescuing GFP::alg-3 transgene is localized in P-granules beginning at the late pachytene stage of male gametogenesis. alg-3/4 double mutants lack a subgroup of small RNAs, named 26G-RNAs, which target and appear to down-regulate numerous spermatogenesis-expressed mRNAs. These findings add to a growing number of AGO pathways required for temperature-dependent fertility in C. elegans and support a model in which AGOs and their small RNA co-factors function to promote robustness in gene-expression networks. Overall design: 3 samples examined. Small RNAs from alg-3(tm1155);alg-4(ok1041);fog-2(q71) males and fog-2(q71) males. Small RNAs from spermatids isolated from ferm-3(q20) worms.
Publication Title
Argonautes ALG-3 and ALG-4 are required for spermatogenesis-specific 26G-RNAs and thermotolerant sperm in Caenorhabditis elegans.
Sample Metadata Fields
Cell line, Subject
View Samples- Mus musculus
- 280 Downloadable Samples
- Illumina HiSeq 2000
Description
Parental dietary conditions can influence the metabolic traits of offspring. In mice, paternal consumption of low protein diet alters cholesterol and lipid metabolism of progeny. Here, we examine RNA species expressed in male reproductive tissues of mice. Protein restriction leads to altered levels of multiple small RNAs in mature sperm, as well as throughout the male reproductive tract, with decreased levels of let-7 family members and increased levels of 5’ fragments of tRNA-Gly isoacceptors. Intriguingly, tRNA fragments are scarce in the testis, but their levels increase in sperm during post-testicular maturation in the epididymis. We find that epididymosomes – extracellular vesicles which fuse with sperm during epididymal transit – exhibit RNA payloads closely matching those of mature sperm, and can deliver tRNA fragments to immature sperm in vitro both in mouse and in bull. Finally, we show that tRNA-Gly-GCC fragments play a role in repressing genes associated with the endogenous retroelement MERVL, both in ES cells and in preimplantation embryos. Our results shed light on small RNA biogenesis during post-testicular sperm maturation, and link tRNA fragments to regulation of endogenous retroelements active in the early embryo. Overall design: IVF was carried out using oocytes from females fed Control diet (C) and sperm from males fed Control diet or Low Protein diet (LP). Zygotes were then developed 2 cell (2C), 4 cell (4C), 8 cell (8C), Morula (M) or Blastocyst (B) embryonic developmental stages when single embryo RNA seq was carried out to study gene expression changes.
Publication Title
Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals.
Sample Metadata Fields
Cell line, Subject
View Samples- Mus musculus
- 187 Downloadable Samples
- NextSeq 500
Description
Parental dietary conditions can influence the metabolic traits of offspring. In mice, paternal consumption of low protein diet alters cholesterol and lipid metabolism of progeny. Here, we examine RNA species expressed in male reproductive tissues of mice. Protein restriction leads to altered levels of multiple small RNAs in mature sperm, as well as throughout the male reproductive tract, with decreased levels of let-7 family members and increased levels of 5’ fragments of tRNA-Gly isoacceptors. Intriguingly, tRNA fragments are scarce in the testis, but their levels increase in sperm during post-testicular maturation in the epididymis. We find that epididymosomes – extracellular vesicles which fuse with sperm during epididymal transit – exhibit RNA payloads closely matching those of mature sperm, and can deliver tRNA fragments to immature sperm in vitro both in mouse and in bull. Finally, we show that tRNA-Gly-GCC fragments play a role in repressing genes associated with the endogenous retroelement MERVL, both in ES cells and in preimplantation embryos. Our results shed light on small RNA biogenesis during post-testicular sperm maturation, and link tRNA fragments to regulation of endogenous retroelements active in the early embryo. Overall design: Zygotes were generated by IVF from animals fed a control diet. These embryos were then microinjected with various combinations of small RNAs and control RNA (HIS3.3::GFP). Follwoing injections the zygotes were developed and allowed to develop until 2 cell (2C) or 4 cell (4C) stage when single embryo RNA seq was carried out to study gene expression changes
Publication Title
Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals.
Sample Metadata Fields
Cell line, Subject
View Samples- Mus musculus
- 103 Downloadable Samples
- NextSeq 500
Description
Parental dietary conditions can influence the metabolic traits of offspring. In mice, paternal consumption of low protein diet alters cholesterol and lipid metabolism of progeny. Here, we examine RNA species expressed in male reproductive tissues of mice. Protein restriction leads to altered levels of multiple small RNAs in mature sperm, as well as throughout the male reproductive tract, with decreased levels of let-7 family members and increased levels of 5’ fragments of tRNA-Gly isoacceptors. Intriguingly, tRNA fragments are scarce in the testis, but their levels increase in sperm during post-testicular maturation in the epididymis. We find that epididymosomes – extracellular vesicles which fuse with sperm during epididymal transit – exhibit RNA payloads closely matching those of mature sperm, and can deliver tRNA fragments to immature sperm in vitro both in mouse and in bull. Finally, we show that tRNA-Gly-GCC fragments play a role in repressing genes associated with the endogenous retroelement MERVL, both in ES cells and in preimplantation embryos. Our results shed light on small RNA biogenesis during post-testicular sperm maturation, and link tRNA fragments to regulation of endogenous retroelements active in the early embryo. Overall design: Zygotes were generated by ICSI from oocytes/females fed a Control diet and sperm/males fed either a Control or Low Protein diet. The sperm was isolated from either the Rete testis or the Cauda epididymis and injected either as a whole sperm or just the sperm head. Following fertilization by ICSI the zygotes developed for 28 hours (2C stage) and were harvested for single-embryo RNA-Seq.
Publication Title
Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals.
Sample Metadata Fields
Cell line, Subject
View Samples- Homo sapiens
- 251 Downloadable Samples
Illumina HumanHT-12 V4.0 expression beadchip
Description
This SuperSeries is composed of the SubSeries listed below.
Publication Title
Integrative epigenome-wide analysis demonstrates that DNA methylation may mediate genetic risk in inflammatory bowel disease.
Sample Metadata Fields
Sex, Age, Specimen part, Subject
View Samples- Homo sapiens
- 251 Downloadable Samples
- Illumina HumanHT-12 V4.0 expression beadchip
Description
Epigenetic alterations may provide important insights into gene-environment interaction in inflammatory bowel disease (IBD). Here we observe epigenome-wide DNA methylation differences in 240 newly-diagnosed IBD cases and 190 controls. These include 439 differentially methylated positions (DMPs) and 5 differentially methylated regions (DMRs), which we study in detail using whole genome bisulphite sequencing. We replicate the top DMP (RPS6KA2) and DMRs (VMP1, ITGB2, TXK) in an independent cohort.
Publication Title
Integrative epigenome-wide analysis demonstrates that DNA methylation may mediate genetic risk in inflammatory bowel disease.
Sample Metadata Fields
Sex, Age, Specimen part
View Samples- Homo sapiens
- 16 Downloadable Samples
- Affymetrix Human Gene Expression Array (primeview), Illumina HumanHT-12 V4.0 expression beadchip, Affymetrix Human Gene 2.0 ST Array (hugene20st)
Description
We present primary results from the Sequencing Quality Control (SEQC) project, coordinated by the United States Food and Drug Administration. Examining Illumina HiSeq, Life Technologies SOLiD and Roche 454 platforms at multiple laboratory sites using reference RNA samples with built-in controls, we assess RNA sequencing (RNA-seq) performance for sequence discovery and differential expression profiling and compare it to microarray and quantitative PCR (qPCR) data using complementary metrics. At all sequencing depths, we discover unannotated exon-exon junctions, with >80% validated by qPCR. We find that measurements of relative expression are accurate and reproducible across sites and platforms if specific filters are used. In contrast, RNA-seq and microarrays do not provide accurate absolute measurements, and gene-specific biases are observed, for these and qPCR. Measurement performance depends on the platform and data analysis pipeline, and variation is large for transcriptlevel profiling. The complete SEQC data sets, comprising >100 billion reads (10Tb), provide unique resources for evaluating RNA-seq analyses for clinical and regulatory settings.
Publication Title
A comprehensive assessment of RNA-seq accuracy, reproducibility and information content by the Sequencing Quality Control Consortium.
Sample Metadata Fields
No sample metadata fields
View Samples