Journal: eLife

Article Title: Base editing strategies to convert CAG to CAA diminish the disease-causing mutation in Huntington’s disease

doi: 10.7554/eLife.89782

Figure Lengend Snippet: ( A ) HEK293 cells were treated with empty vector (EV) or candidate BE strategies such as BE4max-gRNA 1 (gRNA 1) and BE4max-gRNA 2 (gRNA 2) for RNAseq analysis. MiSeq analysis was also performed to judge the levels of CAG-to-CAA conversion. ****, p-value<0.0001 by Student’s t-test (n=4). ( B ) Confirming the lack of significantly altered genes in BE4max-gRNA 1 or BE4max-gRNA 2, we compared all BE-treated samples (n=8) with all EV-treated samples (n=4) to increase the power in the RNAseq differential gene expression analysis. Each circle in the volcano plot represents a gene analyzed in the RNAseq; HTT is indicated by a filled red circle. A red horizontal line represents false discovery rate of 0.05, showing that none was significantly altered by candidate BE strategies. ( C ) We also compared two groups of randomly assigned samples (six samples vs. six samples) to understand the shape of the volcano plot when there were no significant genes.

Article Snippet: Subsequently, genomic DNA for MiSeq analysis and cell pellets for RNAseq analysis were generated from replica plates genome-wide RNAseq analysis (Tru-Seq strand-specific large insert RNAseq) was performed by the Broad Institute.

Techniques: Plasmid Preparation, Gene Expression

Journal: eLife

Article Title: Base editing strategies to convert CAG to CAA diminish the disease-causing mutation in Huntington’s disease

doi: 10.7554/eLife.89782

Figure Lengend Snippet: HEK293 cells were treated with empty vector (EV), or candidate base editing (BE) strategies such as BE4max-gRNA 1 ( A ) and BE4max-gRNA 2 ( B ). Subsequently, DNA samples and RNA samples were collected for MiSeq analysis and RNAseq analysis to evaluate the levels of on-target conversion and changes in transcriptome (n=4), respectively. The most significant gene in cells treated with BE4max-gRNA 2 (panel B) was HSD3B1 , which was not significant by false discovery rate of 0.05 (red lines).

Article Snippet: Subsequently, genomic DNA for MiSeq analysis and cell pellets for RNAseq analysis were generated from replica plates genome-wide RNAseq analysis (Tru-Seq strand-specific large insert RNAseq) was performed by the Broad Institute.

Techniques: Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Developmental and cancer-associated plasticity of DNA replication preferentially targets GC-poor, lowly expressed and late-replicating regions

doi: 10.1093/nar/gky797

Figure Lengend Snippet: Cell line classification based on correlations between replication and gene expression profiles. ( A – C ) Correlation matrices between RFD profiles (C RFD ; A), RNA-seq (C RNA–seq ; B) and MRT profiles (C MRT ; C); Pearson correlation coefficient values are color-coded from blue (0.4) to red (1.0) using the colour bar on the right (Materials and Methods). (Top) A corresponding dendrogram representation of the hierarchical classification of cell lines is shown on top of each correlation matrix; ordinate is the correlation distance (Materials and Methods). ( D ) Cumulative distributions of the absolute MRT changes (|Δ MRT |) between cell lines. Each curve is color-coded according to the pair of cell lines indicated in the insert. The considered threshold of significance (|Δ MRT | >0.2) is indicated by a vertical dotted line.

Article Snippet: Libraries were prepared using the strand specific RNA-Seq library preparation PrepX RNA-seq kit (Wafergen) and a 41 bp paired-end read sequencing was performed on a NextSeq 500 device (Illumina).

Techniques: Expressing, RNA Sequencing Assay