2022 Fiscal Year Research-status Report
Adaptive RNA editing in Cephalopods
| Project/Area Number |
22K15085
|
|---|
| Research Institution | Okinawa Institute of Science and Technology Graduate University |
|---|
Principal Investigator |
|
|---|
| Project Period (FY) |
2022-04-01 – 2024-03-31
|
| Keywords | RNA editing / Cephalopods / Single-cell sequencing |
|---|
| 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.
|
| 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.
|
| Causes of Carryover |
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.
|
