Journal: Nucleic Acids Research

Article Title: Optimized CRISPR-Cas9 system for efficient engineering of ecDNA in cancer cells

doi: 10.1093/nar/gkag005

Figure Lengend Snippet: ecDNA knock-in efficiency of standard and safeguard sgRNAs. ( A ) Schematic showing insertion of a 96-mer TetO repeat sequence into ecDNA of CORL23 cells. See Material and Methods for details of the knock-in system. ( B ) Quantification of CORL23 cell numbers after expression of all-in-one CRISPR plasmids with the indicated sgRNAs and the knock-in template plasmid. Data represent mean ± SD for n = 3 biological replicates. Statistical significance was assessed using a two-sided Student’s t -test. ( C ) Experimental scheme for DNA FISH analyses shown in panels (D–F). (D–F) Box-and-whisker plots showing copy numbers of ( D ) MYC ecDNA and ( E ) TetO knock-in ecDNA in cells transfected with all-in-one CRISPR plasmids with the indicated sgRNAs and the knock-in template plasmid. Panel ( F ) shows the fraction of MYC ecDNA carrying the TetO knock-in sequence. Sample sizes ( n ) are shown below sgRNA labels. Statistical significance was assessed using a Wilcoxon rank-sum test.

Article Snippet: Tracking of indels by decomposition (TIDE) analysis ( https://tide.nki.nl ) was performed using Sanger sequencing traces generated from target-specific PCR products by Eurofins [ ].

Techniques: Knock-In, Sequencing, Expressing, CRISPR, Plasmid Preparation, Whisker Assay, Transfection

Journal: Nucleic Acids Research

Article Title: Optimized CRISPR-Cas9 system for efficient engineering of ecDNA in cancer cells

doi: 10.1093/nar/gkag005

Figure Lengend Snippet: Application of the safeguard sgRNA system in ecDNA-positive CORL23 cells. ( A ) AmpliconArchitect analysis showing the presence of ecDNA structures involving the MYC and PVT1 loci in CORL23 cells.( B ) IGV snapshot showing genome sequence profiles at the MYC and PVT1 loci. ( C ) Representative FISH images of CORL23 cells using an MYC locus probe (red) together with a chromosome 8 probe (green; control for the MYC -containing chromosome). Two representative single cells with different MYC ecDNA copy numbers are shown. Scale bar, 10 µm. ( D ) Schematic illustration of the proposed molecular mechanism by which safeguard sgRNAs fine-tune Cas9 activity. Cytosine extensions decrease Cas9 activity and attenuate Cas9-mediated DNA cleavage via multiple mechanisms, including reduced sgRNA synthesis and impaired target loading efficiency of Cas9–sgRNA RNP complexes (left). When using potent sgRNAs, [C]-extended sgRNAs exhibited decreased bi-allelic indels on chromosomal targets and increased mono-allelic indels in a length-dependent manner (right). This figure was adapted from Kawamata et al. , licensed under the CC BY 4.0. ( E ) Schematic of all-in-one CRISPR plasmids expressing standard and safeguard sgRNAs. ( F ) Western blot analysis of FLAG-tagged Cas9 proteins expressed from the indicated all-in-one plasmids in CORL23 cells. ( G ) Quantification of sgRNA expression levels by RT-qPCR. Left, normalized expression values; right, normalized values multiplied by 1000 and log 2 -transformed. Data represent mean ± SD for n = 3 technical replicates. Statistical significance was assessed using a two-sided Student’s t -test.

Article Snippet: Tracking of indels by decomposition (TIDE) analysis ( https://tide.nki.nl ) was performed using Sanger sequencing traces generated from target-specific PCR products by Eurofins [ ].

Techniques: Sequencing, Control, Activity Assay, CRISPR, Expressing, Western Blot, Quantitative RT-PCR, Transformation Assay

Journal: Cell Genomics

Article Title: Atlas-scale single-cell DNA methylation profiling with sciMETv3

doi: 10.1016/j.xgen.2024.100726

Figure Lengend Snippet: sciMETv3 is compatible with capture methods and enzymatic conversion (A) Experimental design schematic. (B) UMAP of cells combined from both sciMET-cap using bisulfite conversion and non-captured enzymatic conversion preparations. (C) mCH levels show expected patterns for neurons and glial cell populations. (D) Identified clusters with inhibitory and excitatory neuron clusters highlighted. (E) Cluster proportions are comparable between bisulfite + capture and enzymatic non-captured conditions. (F) Global methylation patterns show the expected trend, with cells taken through capture exhibiting lower mCG levels due to the enrichment at regulatory loci with no impact on mCH levels. Box and whiskers represent median and quartiles at each increment (box contains 50%, whiskers contain 75%). (G) Sanger sequencing traces of enzymatic converted libraries show over-conversion of key bases present in the read 2/index read 1 Illumina sequencing primer region that is appended during adapter ligation.

Article Snippet: Box and whiskers represent median and quartiles at each increment (box contains 50%, whiskers contain 75%). (G) Sanger sequencing traces of enzymatic converted libraries show over-conversion of key bases present in the read 2/index read 1 Illumina sequencing primer region that is appended during adapter ligation.

Techniques: Methylation, Sequencing, Illumina Sequencing, Adapter Ligation

Journal: Cell Genomics

Article Title: Atlas-scale single-cell DNA methylation profiling with sciMETv3

doi: 10.1016/j.xgen.2024.100726

Figure Lengend Snippet: sciMETv3 can produce atlas-scale datasets using Illumina or Ultima sequencing platforms (A) Experimental design schematic. (B) Summary of sequencing depth for each platform. Box and whiskers represent median and quartiles. (C) UMAP of Illumina-sequenced cells split by four individuals and colored by cell type. (D) UMAP of Ultima-sequenced cells. (E) Strategy for matching cell coverage distribution between Illumina- and Ultima-sequenced cells for a direct comparison. (F) UMAP of integrated coverage-matched cells from both platforms colored by cell type and split by platform (below). (G) Comparable cell type proportions were achieved for each platform. (H) Comparable global methylation statistics between platforms. Box and whiskers represent median and quartiles.

Article Snippet: Box and whiskers represent median and quartiles at each increment (box contains 50%, whiskers contain 75%). (G) Sanger sequencing traces of enzymatic converted libraries show over-conversion of key bases present in the read 2/index read 1 Illumina sequencing primer region that is appended during adapter ligation.

Techniques: Sequencing, Comparison, Methylation

Journal: Cell Genomics

Article Title: Atlas-scale single-cell DNA methylation profiling with sciMETv3

doi: 10.1016/j.xgen.2024.100726

Figure Lengend Snippet: An atlas of single-cell DNA methylation in human middle frontal gyrus (A) Combined UMAP across both sequencing platforms. (B) Global mCH percentages for the combined dataset. (C) Combined UMAP colored by cell type and split by individual (right). (D) Marker gene body mCH levels by cluster. (E) Cell type proportions across individual and sequencing platforms. (F) mCG levels across marker genes show distinct cluster-specific patterns. (G) Enhancers exhibit highly cell-type-specific hypomethylation compared to promoters.

Article Snippet: Box and whiskers represent median and quartiles at each increment (box contains 50%, whiskers contain 75%). (G) Sanger sequencing traces of enzymatic converted libraries show over-conversion of key bases present in the read 2/index read 1 Illumina sequencing primer region that is appended during adapter ligation.

Techniques: DNA Methylation Assay, Sequencing, Marker

Journal: iScience

Article Title: IFN-γ licenses normal and pathogenic ALPK1/TIFA pathway in human monocytes

doi: 10.1016/j.isci.2024.111563

Figure Lengend Snippet:

Article Snippet: sequence trace decomposition software TIDE , Brinkman et al. , https://tide.nki.nl.

Techniques: Virus, Recombinant, Protease Inhibitor, Transfection, Staining, Reverse Transcription, SYBR Green Assay, Luciferase, Enzyme-linked Immunosorbent Assay, Western Blot, Sequencing, Mutagenesis, Software, Simple Western