| Mitochondrial RNA Transcript Analysis Assay of Arabidopsis Leaf Tissues
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Author:
Date:
2015-10-20
[Abstract] This qPCR-based assay provides an overview of the expression levels of all mitochondrial transcripts (mRNAs and rRNAs) as well as splicing efficiency in Arabidopsis. It was developed before RNAseq techniques were widely used (de Longevialle et al., 2007), but is nevertheless still useful as it is cheaper to run and the analysis is much easier and faster to perform if the aim is only to look at mitochondrial transcripts. For intron-containing mRNAs, the use of primer sets specifically amplifying spliced or unspliced forms allows the evaluation of the splicing efficiency.
[摘要] 这种基于qPCR的测定提供了所有线粒体转录物（mRNA和rRNA）的表达水平以及拟南芥中的剪接效率的概述。 它是在RNAseq技术被广泛使用之前开发的（de Longevialle等人，2007），但是仍然有用，因为它运行更便宜，并且分析更容易和更快地执行，如果目标 只是看看线粒体转录物。 对于含内含子的mRNA，使用特异性扩增剪接或未剪接形式的引物组允许评价剪接效率。
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| Plant Sequence Capture Optimised for Illumina Sequencing
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Author:
Date:
2014-07-05
[Abstract] Plant Sequence Capture is used for targeted resequencing of whole exomes (all exons of a genome) of complex genomes e.g. barley and its relatives (Mascher et al., 2013). Sequencing and computing costs are significantly reduced since only the greatly enriched and gene-coding part of the barley genome is targeted, that corresponds to only 1-2% of the entire genome. Thus, applications such as genetic diversity studies and the isolation of single genes (“cloning-by-sequencing”) are greatly facilitated. Here, a protocol is provided describing the construction of shotgun DNA libraries from genomic barley DNA for sequencing on the Illumina HiSeq/MiSeq systems. The shotgun DNA sequencing libraries are hybridized to an oligonucleotide pool (Exome Library) encompassing the whole ...
[摘要] 植物序列捕获用于复杂基因组（例如大麦及其亲属）的整个外显子（基因组的所有外显子）的靶向重测序（Mascher等人，2013）。测序和计算成本显着降低，因为只有大麦基因组的大量富集和基因编码部分被靶向，其仅对应于整个基因组的1-2％。因此，大大促进了诸如遗传多样性研究和单个基因的分离（"通过测序克隆"）的应用。这里，提供了描述来自基因组大麦DNA的Shotgun DNA文库的构建以在Illumina HiSeq/MiSeq系统上测序的方案。鸟枪DNA测序文库与包含大麦整个外显子组的寡核苷酸池（Exome Library）杂交。外显子组库作为包含生物素化探针（Roche/NimbleGen）的液体阵列提供。随后，使用链霉亲和素包被的磁珠对与Exome文库杂交的基因组鸟枪DNA片段进行亲和纯化。捕获的文库被PCR扩增和测序，使用高通量短读序列合成
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| DNA Methylation Profiling Using Infinium Methylation Assay
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Author:
Date:
2013-05-20
[Abstract] The Infinium Human Methylation 450 BeadChip technology allows the rapid quantitative DNA methylation analysis of more than 485,000 CpG dinucleotides located across the genome. The method utilizes sodium bisulfite treatment of genomic DNA to convert unmethylated cytosine residues into uracils whereas methylated cytosines remain unchanged. Modified DNA is then whole genome amplified, fragmented and hybridized to locus-specific oligomer probes linked to individual beads on a BeadChip. Hybridization is followed by single-base extension of the oligomer with a labeled nucleotide. The BeadChip is subsequently fluorescently stained and scanned to measure the intensities of the beads corresponding to the unmethylated and methylated CpG sites.
[摘要] Infinium人甲基化450 BeadChip技术允许对跨越基因组的超过485,000个CpG二核苷酸进行快速定量DNA甲基化分析。 该方法利用亚硫酸氢钠处理基因组DNA以将未甲基化的胞嘧啶残基转化为尿嘧啶，而甲基化的胞嘧啶保持不变。 然后将修饰的DNA全基因组扩增，片段化并与连接到BeadChip上的单个珠的基因座特异性寡核苷酸探针杂交。 杂交之后是具有标记的核苷酸的寡聚物的单碱基延伸。 随后对珠粒芯片进行荧光染色和扫描，以测量对应于未甲基化和甲基化的CpG位点的珠子的强度。
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