Project/Area Number |
22K15085
|
Research Category |
Grant-in-Aid for Early-Career Scientists
|
Allocation Type | Multi-year Fund |
Review Section |
Basic Section 43050:Genome biology-related
|
Research Institution | Okinawa Institute of Science and Technology Graduate University |
Principal Investigator |
|
Project Period (FY) |
2022-04-01 – 2024-03-31
|
Project Status |
Granted (Fiscal Year 2022)
|
Budget Amount *help |
¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000)
Fiscal Year 2023: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2022: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000)
|
Keywords | RNA editing / Cephalopods / Single-cell sequencing / thermal adaptability |
Outline of Research at the Start |
Cephalopods are poikilotherms and need to regulate different neurophysiological processes at different temperatures. One proposed adaptive mechanism is the A-to-I messenger RNA editing. This project aims to understand how RNA-editing is regulated and the cell-specificity of this mechanism in coleoid cephalopods. The results of this project will unravel how isoforms from different cell types with different RNA-editing levels influence the gene regulatory networks, enabling cephalopods adapt physiologically to the temperature variation.
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Outline of Annual Research Achievements |
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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Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
We have finally collected and isolated cells with good RNA quality and generated cDNA with very good gene coverage.
|
Strategy for Future Research Activity |
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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