Comment[ArrayExpressAccession] E-GEOD-24366 Public Release Date 2010-09-24 Investigation Title Global Changes following N-deprivation in Chlamydomonas: 454 sequencing Comment[Submitted Name] Global Changes following N-deprivation in Chlamydomonas: 454 sequencing Experiment Description Chlamydomonas reinhardtii forms lipid droplets rich in triacylglycerols when nutrient-deprived. To begin studying the mechanisms underlying this process, N-deprivation was used to induce triacylglycerol accumulation and changes in developmental programs such as gametogenesis. Comparative global analysis of transcripts under induced and non-induced conditions was applied as a first approach to study molecular changes that promote or accompany triacylglycerol accumulation in cells encountering a new nutrient environment. Towards this goal, high-throughput sequencing technology was employed to generate large numbers of expressed sequence tags of 8 biologically independent libraries (2 454 libraries, 6 Illumina libraries), four for each condition, N-replete and N-deprived, that allowed a statistically sound comparison of expression levels under the two tested conditions. Inferences on metabolism based on transcriptional analysis can only be indirect but were supported in parts by biochemical experiments. N-deprivation led to a marked redirection of metabolism as the carbon source acetate was no longer converted to cell building blocks by the glyoxylate cycle and gluconeogensis, but funneled directly into fatty acid biosynthesis. Protein biosynthesis and photosynthesis were down-regulated and genes of gametogenesis activated. A notable finding was that the genes encoding photosynthetic accessory PSBS proteins of currently unclear function in C. reinhardtii were strongly expressed following N-deprivation, providing a clue to their physiological role. Lipase-encoding genes were found to be some of the most regulated following N-deprivation, suggesting a remodeling of membranes under these conditions. The data provided here represent a rich source for the exploration of the mechanism of oil accumulation in microalgae. Examing N-replete and N-deprived conditions. One biological replicate each condition. Date of Experiment Term Source Name EFO Term Source Version Term Source File http://www.ebi.ac.uk/efo/efo.owl Person Last Name Wu Benning Person First Name Guangxi Christoph Person Mid Initials Person Email wuguangx@msu.edu Person Affiliation Michigan State University Person Phone Person Fax Person Address Michigan State University, S308 Plant Bio Bld, East Lansing, MI, USA Person Roles submitter Person Roles Term Source REF Person Roles Term Accession Number Normalization Type Normalization Term Accession Number Normalization Term Source REF Replicate Type Replicate Term Accession Number Replicate Term Source REF Experimental Design Experimental Design Term Accession Number Experimental Design Term Source REF Quality Control Type Quality Control Term Accession Number Quality Control Term Source REF Protocol Name P-GSE24366-1 P-GSE24366-3 P-GSE24366-2 P-GSE24366-4 Protocol Description For N-replete growth, TAP medium (Harris, 2009a) with 10 mM NH4+ (TAP+N) was used. N-deprivation was defined as growth in TAP+N to 5x10^6 cells/mL, followed by transfer to TAP-N for 48 hours. To generate material for high-throughput sequencing, cells were grown in 100 TAP+N to 5x10^6 cells/mL. The cultures were split in half and cells were collected by centrifugation, with one pellet being resuspended in 50 mL TAP+N, and the other in 50 mL TAP-N. After 48 hours, the total RNA was harvested using a QIAGEN RNeasy Plant Mini kit (QIAGEN, Valencia, CA, USA). The RNA samples were treated with QIAGEN RNase-free DNase I during extraction. Full-length cDNA pools were generated with the Clontech SMART cDNA library construction kit (Clontech, Mountain View, CA, USA). cDNA was synthesized using a modified cDNA synthesis primer (5M-bM-^@M-^YTAGAGACCGAGGCGGCCGACATGTTTTGTTTTTTTTTCTTTTTTTTTTVN3M-bM-^@M-^Y). Full-length cDNAs were amplified by PCR and pooled to increase their concentration. An SfiI digest was performed, followed by size fractionation. Fractions with the highest intensity and size distribution were pooled and purified. The resulting cDNA pools were then submitted to the MSU-Research Technologies Service Facility (RTSF) for sequencing on a 454 GSFLX Titanium Sequencer (454 Life Sciences, Branford, CT, USA). The cells were grown in liquid cultures under continuous light (~80 M-5mole photons m-2 s-1). For 454 data, default parameters were used to pass reads using 454 quality control tools. The filtered 454 sequencing reads were mapped to the C. reinhardtii v4.0 assembly from the Joint Genome Institute with GMAP (Wu and Watanabe, 2005). In GMAP, the maximum intron length was set at 980bp, which is at the 95 percentile of annotated C. reinhardtii intron lengths. Protocol Software Protocol Hardware Protocol Contact Protocol Type specified_biomaterial_action nucleic_acid_extraction grow feature_extraction Protocol Term Source REF Protocol Term Accession Number Experimental Factor Name CONDITION Experimental Factor Type condition Experimental Factor Term Source REF Experimental Factor Term Accession Number Publication Title Changes in transcript abundance in Chlamydomonas reinhardtii following nitrogen deprivation predict diversion of metabolism. Publication Author List Miller R, Wu G, Deshpande RR, Vieler A, GM-CM-$rtner K, Li X, Moellering ER, ZM-CM-$uner S, Cornish AJ, Liu B, Bullard B, Sears BB, Kuo MH, Hegg EL, Shachar-Hill Y, Shiu SH, Benning C PubMed ID 20935180 Publication DOI 10.1104/pp.110.165159 Publication Status Publication Status Term Source REF Publication Status Term Accession Number Comment[SecondaryAccession] GSE24366 SRP003629 Comment[AdditionalFile:Data1] GSE24366_readme.txt Comment[GEOReleaseDate] 2010-09-23 Comment[GEOLastUpdateDate] 2012-03-22 Comment[AEExperimentType] transcription profiling by high throughput sequencing Comment[ArrayExpressSubmissionDate] 2010-09-23 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR066640-SRR066641 SDRF File E-GEOD-24366.sdrf.txt