Single-cell RNA sequencing of tobacco Bright Yellow-2 cell line reveals heterogeneity in cell cycle phase
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PROVIDER: PRJNA795047 | ENA |
REPOSITORIES: ENA
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Project description:Cell proliferation and division are fundamental processes of plant development and homeostasis. With the feature of high homogeneity, tobacco Bright Yellow-2 (BY-2) cells, a so-called “HeLa” cell line in plants, provide a useful system for study the cell cycle of plants, particularly when in combination with the latest high-throughput single-cell RNA sequencing (scRNA-seq) technology. Here, we generated both scRNA-seq and bulk RNA-seq data from the BY-2 cells and found that the enzymatic hydrolysis during protoplasting was the major factor contributing to cell clustering noise. We introduced the ppScoring concept to filter out confounding cells due to protoplasting and clustered the remaining cells into clusters corresponding to G0-G1, S, and G2-M phases as well as clusters with phase transition arrested or cell cycle exit cells. The clustering results were supported by cell cycle phase specific marker genes and reconstruction of the continuous full cell cycle phases, i.e., from G0-G1 to S to G2-M, based on pseudotime trajectory analysis. The clustering results also identified a set of 896 cell cycle marker genes. This work demonstrated cell cycle-dependent transcriptional heterogeneity of the BY-2 cell population, provided marker genes for study of the cell cycle of plants and new insights into the progression of cell division.
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Project description:Tobacco BY-2 cells can be highly synchronized by treatment with an inhibitor of DNA synthesis, aphidicolin. For comprehensive understanding of cell cycle-regulated gene expression in tobacco, microarray analysis was conducted in synchronized cultures of BY-2 cells. Cells were treated with aphidicolin for 24 hours to block the entry into and progression through the S phase. After release from the aphidicolin block, cells were sampled every 2 h from 0–16 h. cDNA probes prepared from these samples were hybridised to a custom-made16K microarray. We performed this procedure twice independently to identify transcripts that reproducibly showed similar cell cycle-regulated expression. A total of 883 putative cell cycle-regulated transcripts were identified from 16,896 duplicated spots on the microarrays. These transcripts could be categorised into 10 classes on the basis of transcript expression patterns in the cell cycle. Keywords: time course
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Project description:Tobacco BY-2 cells can be highly synchronized by treatment with an inhibitor of DNA synthesis, aphidicolin. For comprehensive understanding of cell cycle-regulated gene expression in tobacco, microarray analysis was conducted in synchronized cultures of BY-2 cells. Cells were treated with aphidicolin for 24 hours to block the entry into and progression through the S phase. After release from the aphidicolin block, cells were sampled every 2 h from 0â16 h. cDNA probes prepared from these samples were hybridised to a custom-made16K microarray. We performed this procedure twice independently to identify transcripts that reproducibly showed similar cell cycle-regulated expression. A total of 883 putative cell cycle-regulated transcripts were identified from 16,896 duplicated spots on the microarrays. These transcripts could be categorised into 10 classes on the basis of transcript expression patterns in the cell cycle. Keywords: time course Synchronization experiments were repeated twice (experiments A and B). In each experiment, cells were sampled every 2 h during the culture periods (0-16 h), and used for microarray analysis.
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Project description:Eleven healthy human subjects were enrolled in a 6-day simulated shift work protocol. Blood samples were collected during the two 24-hour measurement periods. Blood samples were collected every 4 hours during both measurement periods. Subjects entered the lab on Day 1. At the start of Day 2, the first 24-hour measurement period was started. Subjects slept according to their habitual sleep/wake schedule, followed by a 16-hour constant posture procedure. On days 3-6, the sleep period was delayed by 10 hours. Participants in the control group were exposed to dim light throughout the waking periods; participants in the bright group were exposed to bright light (~6,000lux) for 8 hours during the waking period. Following the third night on this schedule, subjects underwent another 24-hour measurement period. During both measurement periods, blood samples were collected and PBMCs were isolated. mRNA was extracted, labelled, and hybridized to microarrays.
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