Project description:Single cell mRNA-seq (3' UMI counting) experiments of the sperm and vegetative nuclei from the Arabidopsis pollen to investigate the heterogeneity of those cell types.
Project description:The formation of a zygote by the fusion of egg and sperm involves the two gametic transcriptomes. In flowering plants, the embryo sac embedded within the ovule contains the egg cell, while the pollen grain contains two sperm cells inside a supporting vegetative cell. The difficulties of collecting isolated gametes and consequent low recovery of RNA have restricted in-depth analysis of gametic transcriptomes in flowering plants. We isolated living egg cells, sperm cells, and pollen vegetative cells from rice, and identified transcripts for ~36,000 genes by deep sequencing. The three transcriptomes are highly divergent, with about three quarters of those genes differentially expressed in the different cell types. Distinctive expression profiles were observed for genes involved in chromatin conformation, including an unexpected expression in the sperm cell of genes associated with active chromatin. Furthermore, both the sperm cell and the pollen vegetative cell were deficient in expression of key RNAi components. Differences in gene expression were also observed for genes for hormonal signaling and cell cycle regulation. The egg cell and sperm cell transcriptomes reveal major differences in gene expression to be resolved in the zygote, including pathways affecting chromatin configuration, hormones and cell cycle. The sex-specific differences in expression of RNAi components suggest that epigenetic silencing in the zygote might act predominantly through female-dependent pathways. More generally, this study provides a detailed gene expression landscape for flowering plant gametes, enabling the identification of specific gametic functions and their contributions to zygote and seed development. The gene expression profiles of the three gametic cells, egg cell (EC), sperm cell (Sp) and vegetative cell (Ve) were compared via RNA-seq using three biological replicates for each. Differential expression (DE) analysis was performed using edgeR and the resulting DE genes were assessed for Gene Ontology term enrichment.
Project description:The Arabidopsis thaliana central cell, the companion cell of the egg, undergoes DNA demethylation prior to fertilization, but the targeting preferences and biological significance of this process remain unclear. Here, we show that active DNA demethylation mediated by the DEMETER DNA glycosylase accounts for all of the demethylation in the central cell, and preferentially targets small, AT-rich and nucleosome-depleted transposable elements. The vegetative cell, the companion cell of sperm, also undergoes DEMETER-dependent demethylation of similar sequences, and lack of DEMETER in vegetative cells causes reduced small RNA-directed DNA methylation of transposons in sperm. Our results demonstrate that demethylation in companion cells reinforces transposon methylation in plant gametes, thereby assuring stable silencing of transposable elements across generations Examination of DNA methylation in Arabidopsis endosperm, embryo, and pollen
Project description:We have been performing single-cell RNAseq profiling for the entire adult Drosophila. Here we are providing raw data generated from the Smart-seq2 platform. They are from 37 384-well plates.
Project description:Epigenetic inheritance is more widespread in plants than in mammals, in part because mammals erase epigenetic information each generation by germline reprogramming. To assess the extent of germline reprogramming in plants, we sequenced the methylome of three haploid cell types from developing pollen: the sperm cell (SC), the vegetative cell, and their precursor the post-meiotic microspore. Whole genome bisulfite sequencing of FACS-purified sperm cells, vegetative nuclei and microspores
Project description:We developed a transcriptome resource for Douglas-fir covering key developmental stages of megagametophytes over time: prefertilization, fertilization, embryogenesis, and early, unfertilized abortion. Extracted RNA was sequenced using large-scale sequencing and reads were assembled to generate a de novo reference transcriptome of 105,505 predicted high-confidence transcripts. Expression levels were estimated based on alignment of the original reads to the reference. 200â400 megagametophytes were dissected and pooled per sample on four dates from either pollinated or unpollinated cones: June 10, June 22, June 30, and July 6 2011. These dates coincided with key events in seed development: corrosion cavity formation, fertilization, embryogenesis, or the early stages of abortion in the unpollinated treatment. Sporophytic tissue (i.e. cone bracts and cone scales) were added for comparison. PolyA RNA was used for Illumina sequencing.