| 研究課題/領域番号 |
22K15085
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| 研究機関 | 沖縄科学技術大学院大学 |
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研究代表者 |
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| 研究期間 (年度) |
2022-04-01 – 2024-03-31
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| キーワード | RNA editing / Cephalopods / Single-cell sequencing |
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| 研究実績の概要 |
We have applied different single-cell RNA sequencing technologies in neural squid organs. One of these technologies uses a combination of 10X genomics and long-read sequencing with ONT or PacBio, from which we observed isoform-specific and RNA-editing at the 3' end corresponding to a cell type. However, the gene coverage and gene content per cell were still very low. We also observed that the highly repetitive genome of cephalopods limits the sequencing of entire gene isoforms and generates shorter sequences and significant levels of molecules coming from strand-invasion patterns. We have started sequencing single cells using plate-based sorting to solve our current limitations. Using our Euprymna berryi genome, I modified TSO-UMI primers to reduce the number of molecules with strand invasion. As a result, we have observed much larger cDNA peaks than in the previous technologies, indicating a successful fresh cell collection and skipping molecules with strand invasion during the RT-PCR.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
1: 当初の計画以上に進展している
理由
We have finally collected and isolated cells with good RNA quality and generated cDNA with very good gene coverage.
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| 今後の研究の推進方策 |
We have started sequencing single cells using plate-based sorting to solve our current limitations. Using our Euprymna berryi genome, I modified TSO-UMI primers to reduce the number of molecules with strand invasion. As a result, we have observed much larger cDNA peaks than in the previous technologies, indicating a successful fresh cell collection and skipping molecules with strand invasion during the RT-PCR.
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| 次年度使用額が生じた理由 |
The season, lifespan, and spawning time of our species limited us to collected enough individuals for our experiment at the correct time of the first-fiscal year. We, however, generated preliminary data using the available animals.
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