sanger sequence traces  (Synthego Inc)

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: Developmental cell

Article Title: Cues from mRNA splicing prevent default Argonaute silencing in C. elegans

doi: 10.1016/j.devcel.2021.08.022

Figure Lengend Snippet: (A) Schematic structures of intron-containing cdk-1::gfp and intronless cdk-1*::gfp* transgenes. (Gray boxes, cdk-1 coding sequence; green boxes, gfp coding sequence; V-shaped lines, introns).

Article Snippet: Sanger sequencing trace files , This paper , Mendeley Data: https://doi.org/10.17632/c8h2f2prch.2.

Techniques: Sequencing

Journal: Developmental cell

Article Title: Cues from mRNA splicing prevent default Argonaute silencing in C. elegans

doi: 10.1016/j.devcel.2021.08.022

Figure Lengend Snippet: (A) Schematic outlining the removal of endogenous oma-1 coding region and insertion of intron-containing oma-1 or intronless oma-1* alleles. CRISPR was used to modify the endogenous oma-1 locus. An in-frame 3xflag sequence was first inserted at the 3′ end of the oma-1 gene. The oma-1 gene from the middle of exon 1 to the end of exon 6 was replaced with a short stuffer containing a new CRISPR guide site (marked with a black dashed rectangle) to allow subsequent insertion of the wild-type oma-1 sequence with or without (oma-1*) introns. Silencing of intronless oma-1* was confirmed by oma-2(RNAi). To test whether intronless oma-1* is also silenced by the cis-silencing pathway, CRISPR was used to delete rde-3 and then oma-2. The oma-2 genotype of F2 progeny (n = 48) was determined by PCR. The percentage of each genotype is indicated, and the percentage of fertile or sterile worms is indicated. Every CRISPR gene editing event was validated by genomic PCR and Sanger sequencing. (B and C) Plots showing the density of antisense small RNAs mapping along the coding regions of (B) oma-1::3xflag or (C) oma-1*::3xflag. Positions of exon junctions in oma-1::3xflag (and corresponding locations in intronless oma-1*) are indicated by broken vertical lines in gene cartoon. The height of each bar represents the number of reads that begin at that position per million total reads.

Article Snippet: Sanger sequencing trace files , This paper , Mendeley Data: https://doi.org/10.17632/c8h2f2prch.2.

Techniques: CRISPR, Sequencing, Sterility

Journal: Developmental cell

Article Title: Cues from mRNA splicing prevent default Argonaute silencing in C. elegans

doi: 10.1016/j.devcel.2021.08.022

Figure Lengend Snippet: (A) Schematic gene structures of intron-containing gfp::his-61 (left) and intronless gfp*::his-61 (right), and representative epifluorescence images analyzing GFP expression in embryos (soma), oocytes, or distal tips of dissected gonads (outlined with white dashes in gfp*::his-61). CRISPR was used to delete rde-3(ne4848) to test the role of RDE-3 in silencing intronless gfp*::his-61. In the resulting rde-3 mutant, epifluorescence (upper) and DIC (lower) imaging of a representative gonadal arm shows GFP signal in the distal germline. GFP signal gradually decreases in the pachytene region and is absent in oocytes. Percent GFP+ (ON) or GFP− (OFF) worms and the number of worms analyzed is indicated. Every CRISPR gene-editing event was validated by somatic GFP expression (in cases of gfp integration), genomic PCR and Sanger sequencing.

Article Snippet: Sanger sequencing trace files , This paper , Mendeley Data: https://doi.org/10.17632/c8h2f2prch.2.

Techniques: Expressing, CRISPR, Mutagenesis, Imaging, Sequencing

Journal: Developmental cell

Article Title: Cues from mRNA splicing prevent default Argonaute silencing in C. elegans

doi: 10.1016/j.devcel.2021.08.022

Figure Lengend Snippet: (A and B) Plots showing the density of antisense small RNAs mapping along intronless oma-1*::gfp* transgene in the (A) wild-type or (B) oma-1 deletion worms. The schematic in (B) outlines the deletion of endogenous oma-1 (including promoter and 30 UTR) on chromosome IV (LGIV), single-copy insertion of intronless oma-1*::gfp* on LGII, and small RNA sequencing. oma-1 deletion at the endogenous locus was validated by genomic PCR and Sanger sequencing analyses. (C) Model figure. Introns/splicing protect transcripts from default WAGO-dependent and cis-silencing. (Top) Factors associated with splicing (orange ovals) counteract silencing cues (red octagons) deposited on pre-mRNA by default. (Bottom) Silencing cues on unspliced transcript recruit RdRP, which makes small RNAs that guide Argonaute-mediated trans-silencing of cognate intron-containing genes (dashed arrow). In addition, possibly in response to the same cues, unspliced transcripts are silenced in cis, for example, by disrupting mRNA processing or promoting export to nuage where RNAs are sequestered and used for small-RNA templating.

Article Snippet: Sanger sequencing trace files , This paper , Mendeley Data: https://doi.org/10.17632/c8h2f2prch.2.

Techniques: RNA Sequencing, Sequencing

Journal: Developmental cell

Article Title: Cues from mRNA splicing prevent default Argonaute silencing in C. elegans

doi: 10.1016/j.devcel.2021.08.022

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Sanger sequencing trace files , This paper , Mendeley Data: https://doi.org/10.17632/c8h2f2prch.2.

Techniques: RNA Sequencing, Sequencing, Recombinant, Microscopy, Reverse Transcription, SYBR Green Assay, Isolation, CRISPR, Software