| 研究実績の概要 |
* 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.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
4: 遅れている
理由
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.
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