GNPS_GC_3D_skin_mapping_longitudinal_high_res_min
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Agilent 7200 GC-QTOF
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Pieter Dorrestein
PROVIDER: MSV000084338 | GNPS |
REPOSITORIES: GNPS
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Project description:All of the sampling has been conducted under UC San Diego IRB #171662. Cleaned and degassed PDMS patches have been placed on skin of a volunteer overnight and secured in place with waterproof bandage. After sampling, the bandage was removed, patches placed into 2 mL borosilicate vials using clean tweezers and 150 uL of HPLC grade methanol added for extraction. The extraction was carried out overnight at room temperature. After extraction, the sample was transferred into a 200 uL insert in a 2 mL vial and the vial capped with a crimp camp with silicone septum. The GC-MS analysis was carried out using the Agilent 7200 GC/QTOF equipped with robotic sampler system.
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Approximately 50 mg of sample were placed in 2 ml borosilicate vials capped with a screw cap with silicone septum. The capped vials were stored at -20 C and thawed immediately prior to analysis. The GC-MS analysis was carried out using the Agilent 7200 GC/QTOF equipped with robotic sampler system. The volatiles from the sample were extracted from headspace using Polydimethylsiloxane/Divinylbenzene (PDMS/DVB) df 65 um Solid Phase Microextraction Fiber (SPME) for 30 minutes at 50 C. The fiber was then inserted into the injector equipped with Merlin septum heated to 250 C and the adsorbed compounds were desorbed for 1 minute.
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Project description:Development of the soil amoeba Dictyostelium discoideum is triggered by starvation. When placed on a solid substrate, the starving solitary amoebae cease growth, communicate via extracellular cAMP, aggregate by tens of thousands and develop into fully integrated multicellular organisms. Early phases of the developmental program are often studied in cells starved in suspension while cAMP is provided exogenously. Previous studies revealed massive shifts in the transcriptome under these developmental conditions and a close relationship between gene expression and morphogenesis, but were limited by the sampling frequency and the resolution of the methods. Here, we combine the superior depth and specificity of RNA-seq with high frequency sampling during filter development and cAMP pulsing in suspension. We found that the developmental transcriptome exhibits mostly gradual changes interspersed by a few instances of large changes. Considering the whole transcriptome as a single phenotype, we treated each time point as an independent entity and were able to characterize development as groups of similar time points separated by gaps. The grouped time points represent gradual changes in mRNA abundance and the gaps represent times during which many genes are differentially expressed rapidly. Comparing development on solid substrate to development in suspension revealed that gene expression in filter developed cells lagged behind cells treated with exogenous cAMP in suspension. The high sampling frequency revealed many genes whose regulation is reproducibly more complex than indicated by previous studies. Gene Ontology enrichment analysis showed that the transition to multicellularity at the mound stage coincided with rapid accumulation of transcripts associated with mitosis and DNA replicative processes. Later development included the up-regulation of organic signaling molecules and co-factor biosynthesis. We observed multiple instances of enrichment of oxidation-reduction and reactive oxygen related terms. Our analysis also demonstrated a high level of synchrony between the developing structures throughout development. Our data describe D. discoideum development as a series of coordinated cellular and multicellular activities. Coordination occurred within fields of aggregating cells and between multicellular bodies, such as mounds or migratory slugs that experience both cell-cell contact and various soluble signaling regimes. These time courses, sampled at the highest temporal resolution to date in this system, provide a comprehensive resource for future studies of developmental gene expression. Dictyosteloium discoideum gene expression (mRNA) profiles from two experimental time courses: development on filters and cAMP signaling in suspension. Filter development consists of 2 replicates of 19 time point samples, while cAMP in suspension contains 3 replicates of 10, 10 and 9 samples each.
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