Transcriptional_profiling_of_Chlamydomonas_reinhardtii. Transcriptional_profiling_of_Chlamydomonas_reinhardtii

In this study we are using RNA-Seq, a recently developed approach to transcriptome profiling that uses deep-sequencing technologies, to catalogue all of the transcripts and quantify the changing expression levels during gametogenesis. Previous study shows Chlamydomonas and Plasmodium share similar fertilization process. The protein HAP2/GCS1, which is expressed specifically on the surface of sperm cells, was identified in both Chlamydomonas and Plasmodium from homologies to sperm cell proteins isolated from Lilium (lily) generative cells. The loss-of-function mutations in HAP2/GCS1 prevent gamete fusion but the hap2/gcs1 mutant sperm cells are able to adhere with the female gametes. These results suggest that HAP2 protein is essential for gamete fusion, the last step of fertilization. Therefore, a conserved mechanism for fertilization is used in both species. The unicellular biflagellated green algae Chlamydomonas reinhardtii is an excellent model system to dissect the sexual cell–cell recognition and diploid formation process morphologically and physiologically. Chlamydomonas reinhardtii is sexually heterothallic, with separate mt+ and mt- strains. Cells of both mating types independently differentiate from vegetative cells to gametes under nitrogen-starved conditions. In this study poly(A)+ RNA from vegetative cells, mt+ gametes and mt- gametes were isolated and used to produce double-stranded cDNA. Then the double-stranded cDNA was sent to Sanger Institute for RNA-seq for the first run. And for quanlity control, double-stranded cDNA of mt- gametes were synthesized with oligo dT or random 9mer primers for the secondary run. Protocols. Sample Preparation and cDNA Library Construction - Chlamydomonas reinhardtii strains 21gr (mt+) (CC-1690), 6145C (mt-) (CC-1691 are available from the Chlamydomonas Genetics Center, Duke University. Unsynchronized vegetative cells were grown under continuous light in liquid M-medium, synchronized vegetative cells were cultured with aeration at 23°C on a 13:11 h light/dark cycle in M-medium, and synchronized vegetative cells were induced to become gametes by incubation in medium without nitrogen (N-free medium) followed by culturing in continuous light with aeration at room temperature for 17 h and the cells were harvested at 4 h after dark-light switch. Lysin-treated gametes were gametes pellet resuspended with lysin for 1hr, activated gametes were activated with db-cAMP for 1 h. Samples were pelleted and resuspended in lysis buffer(50mM Tris-Cl: pH 7.5, 150mM NaCl, 15mM EDTA, 2% SDS, 40ug/ml Proteinase K). Following organic extraction (25:24:1 [v/v] phenol/chloroform/isoamyalcohol), RNA was recovered by precipitation with equal volume of isopropanol. Total RNA resuspended in water was further purified with TRIzol Reagent (Invitrogen) following the manufacturer’s manual. mRNA was extracted from total RNA using Dynabeads Oligo(dT) (Invitrogen Dynal) following the manufacturer's instuctions. After elution from the beads, first- and second-strand cDNA was generated using Just cDNA Double-Stranded cDNA Synthesis Kit (Agilent) following the manufacturer's directions. Treatment Groups - Total RNA was prepared from eight differing treatment groups: synchronized vegetative (V-syn), non-synchronized vegetative (V-asyn), minus-gametic (MG), activated minus-gametic (MG-A), lysin-treated minus-gametic (MG-L), plus-gametic (PG), activated plus-gametic (PG-A), and lysin-treated plus-gametic (PG-L) cells. Briefly, V-syn cells were harvested at mid-day and checked for flagellation, indicative of cell cycle exit. V-asyn cells, however, were grown under continuous light, and thus should include transcripts representing both flagellated and de-flagellated states. MG or PG cells were obtained by starving vegetative cells of nitrogen for a fortnight, followed by harvest the following afternoon. MG-A or PG-A cells were generated via exposure of db-cAMP to MG or PG cells, respectively, for an hour, followed by immediate harvest. To approximate physiological lysin exposure, MG-L or PG-L cells were obtained by exposing for an hour, either MG or PG cells, to lysin-containing supernatant purified from activated gamete pools. For each treatment group, a sample size of three biological replicates was prepared. Transcriptome Preparation & Sequencing - Oligo-dT primers were used to enrich mRNA species from complex total RNA pools, and cDNA was subsequently synthesized using random primers. Complex cDNA representing the transcriptomes of each of the eight treatment groups were sequenced individually on HiSeq machines. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/