Laboratory for Functional Genomics, Nutrigenomics and Systems Biology, Scientific Genomics Platforms, Max Delbrück Center for Molecular Medicine/Berlin Institute of Health, 13092, Berlin, Germany.
Commun Biol. 2020 Aug 14;3(1):439. doi: 10.1038/s42003-020-01167-x.
In vitro transcription using T7 bacteriophage polymerase is widely used in molecular biology. Here, we use 5'RACE-Seq to screen a randomized initially transcribed region of the T7 promoter for cross-talk with transcriptional activity. We reveal that sequences from position +4 to +8 downstream of the transcription start site affect T7 promoter activity over a 5-fold range, and identify promoter variants with significantly enhanced transcriptional output that increase the yield of in vitro transcription reactions across a wide range of template concentrations. We furthermore introduce CEL-Seq , which uses an optimized T7 promoter to amplify cDNA for single-cell RNA-Sequencing. CEL-Seq+ facilitates scRNA-Seq library preparation, and substantially increases library complexity and the number of expressed genes detected per cell, highlighting a particular value of optimized T7 promoters in bioanalytical applications.
使用 T7 噬菌体聚合酶进行体外转录在分子生物学中被广泛应用。在这里，我们使用 5'RACE-Seq 来筛选 T7 启动子的随机初始转录区域，以检测与转录活性的串扰。我们揭示了转录起始位点下游的+4 到+8 位置的序列在 5 倍的范围内影响 T7 启动子的活性，并鉴定出具有显著增强转录输出的启动子变体，这些变体可以在广泛的模板浓度范围内提高体外转录反应的产量。我们进一步引入了 CEL-Seq，它使用优化的 T7 启动子来扩增 cDNA 进行单细胞 RNA-Seq。CEL-Seq+ 促进了 scRNA-Seq 文库的制备，并显著增加了文库的复杂性和每个细胞检测到的表达基因的数量，这突出了优化的 T7 启动子在生物分析应用中的特殊价值。
