2014 Fiscal Year Annual Research Report
Single cell transcriptome analysis
| Project/Area Number |
25710018
|
|---|
| Research Institution | The Institute of Physical and Chemical Research |
|---|
Principal Investigator |
PLESSY Charles 国立研究開発法人理化学研究所, ライフサイエンス技術基盤研究センター, ユニットリーダー (60391984)
|
|---|
| Project Period (FY) |
2013-04-01 – 2016-03-31
|
| Keywords | 生物学 / ハイスループット / トランスクリプトーム / 単一細胞 |
|---|
| Outline of Annual Research Achievements |
* We continued our optimisations of the nanoCAGE protocol. In particular, we made a significant improvement to the step where sequencing adapters were added. Previously, this was done by a standard PCR using primers with long tails carrying the adaptors, and the minimum starting materials was 80 ng of cDNA, which had to be obtained by a strong PCR pre-amplification. In the improved protocol, we use the Tn5 transposase "tagmentation" system, with as few as 0.25 ng of cDNA as starting material. We could therefore considerably reduce the number of cycles of the pre-amplification PCR, and therefore reduce biases, noise and failure rates.
* The experiments for the development of special reverse-trancription oligonucletides that deplete adaptor artefacts and ribosomal sequences, and that we termed "pseudo-random" primers, were completed successfully. We tested these pseud-random primers on single cells and decided to use them instead of the standard random primers.
* For the in silico detection of cell cycle phases, we completed the quality controls on the RNA-seq and cell images taken from single HeLa Fucci cells the Fluidigigm C1 platform for single-cell analysis.
|
| Current Status of Research Progress |
Current Status of Research Progress
4: Progress in research has been delayed.
Reason
The delay accumulated in FY H25, that happened mostly because it took 6 months to recruit a post-doctoral scientist, was not recovered in FY H26, and therefore was carried over to FY H27.
|
| Strategy for Future Research Activity |
The plan for FY H27 was to make final optimisations and controls of our "nanoCAGE" protocol and then prepare a large-scale data set of single-cell transcriptomes sequenced on the HiSeq platform.
|
| Causes of Carryover |
Since hiring could only be done in November 2013, there was a carry-over from FY2013 to FY2014, which was again carried over to FY2015.
|
| Expenditure Plan for Carryover Budget |
Personnel Expenditure and Remuneration.
|
