2013 Fiscal Year Annual Research Report
Single cell transcriptome analysis
Project/Area Number |
25710018
|
Research Institution | Institute of Physical and Chemical Research |
Principal Investigator |
PLESSY Charles 独立行政法人理化学研究所, 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 – 2015-03-31
|
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 / 幹細胞 |
Research Abstract |
The goal of our project is to quantify the expression levels of genes by transcriptome sequencing in single cells, to better understand the biology of embryonic stem cells, and contribute to the field of regenerative medicine. * We have started this project by improving the efficiency of our method for gene expression analysis (nanoCAGE), so that it can be used on single cells. We tested and identified more efficient enzymes for the amplification steps by PCR. We also developed an original modification of our reverse-transcription oligonucleotides so that their random part, which primes the synthesis of cDNAs, is depleted from combinations that can cause the formation of artefacts by cross-hybridisation of the primers. In parallel, we have established the appropriate conditions for isolating single cells at a high throughput with a flow cytometer. * For the large-scale analysis of the data, we have developed an automated processing workflow and tools, that researchers and technicians can apply by themselves to data freshly sequenced, without needing the help of a bioinformatician for drawing the first conclusions of an experiment (did it work ? Is there a difference between the treatment and the control ? Is gene X expressed in the cells ?). * We also have produced a pilot reference set of single HeLa cells taken at different phases of the cell cycle, using Fluidigm C1 machines. We will use it to assess our first large-scale dataset, that we are currently planning on embryonic stem cells isolated with a flow cytometer.
|
Current Status of Research Progress |
Current Status of Research Progress
4: Progress in research has been delayed.
Reason
1) The original plan included the participation of a post-doctoral researcher. However the recruitment took time and the recruited researcher started on November 1st, which is a half-year delay compared to the ideal situation. 2) Prior the arrival of the post-doctoral researcher, some of the planned work was done by a technical scientist. Unfortunately, he has quit for personal reason at the end of June, and this also delayed the project. 3) We have spent more time modifying the nanoCAGE protocol, to take advantage of a new tool from Illumina/Nextera, called "tagmentation", which we expect to bring dramatic improvements: with tagmentation, only 1 ng of PCR product is needed to prepare cDNAs for sequencing, in comparison with 80 ng for the current nanoCAGE protocol. While this improvement will benefit to this project, it also delayed it of a few months for the production of our first large-scale dataset of ES cells. Given the delay caused by personnel recruitment and replacement, it is possible that we will ask for an extension of this grant to FY2015, since the delay in recruitment also caused a delay in spending the personnel budget, reflected by a strong carry-over from FY2013 to FY2014.
|
Strategy for Future Research Activity |
* We are concluding our experiments on improving the nanoCAGE protocol, and will then produce single-cell transcriptomes by batches of 1,000 in 96-well plates. * Once this milestone is reached, we will start the development of a method for directly sequencing the first-strand cDNAs from single cells without PCR amplification. The progresses on the improved nanoCAGE protocol will directly benefit to the development of this non-amplified method. * In parallel, we are collaborating with the Fujii laboratory at the Institute of Industrial Sciences, University of Tokyo. We have finalised the design and production of a microfluidics device to encapsulate single cells in microdroplets, where the cells are lysed and their RNAs converted to cDNAs. Our next step is to use the device to produce pilot data to publish this proof of concept. * For the single-cell data analysis, we are working with the bioinformatics group of our research division. Our main developments are the on interactive visualisation, both as a tool for analysis and as a tool for explaining our work to the public, and on classification of cell transcriptomes to determine cell types.
|
Expenditure Plans for the Next FY Research Funding |
We carried over some fund from FY2013 to FY2014 because we could not hire until November 2013, with strongly reduced spending for the first 8 months. In fiscal year 2014, we need: one year of salary for a post-doctoral researcher, travel money for one conference, enzymes, oligonucleotides and other reagents for preparing transcritpome libraries, and kits for quantitative sequencing on the Illumina platform. Construction of the libraries will take place at the beginning of the fiscal year, sequencing will take place during summer, and participation to an international conference will take place during the second half of the fiscal year.
|