dynamicarray 96 96 chips  (fluidigm)

Journal: Nucleic Acids Research

Article Title: CRISPR-Cas9 targeting of G-Quadruplex DNA in ADH1 promoter highlights its role in transcriptome and metabolome regulation

doi: 10.1093/nar/gkaf853

Figure Lengend Snippet: G4 CRISPR-Cas9 strategy to mutate the G4 motif at adh1 + promoter in S. pombe . ( A ) Schematics illustrating important steps for mutating Adh1 G4 motif using G4 CRISPR-Cas9 system in S. pombe . In short, sgRNAs targeting simultaneously different sites within the G4 motif were designed and Golden Gate Cloning that allows scarless cloning in a single step was used to insert the sgRNAs into a plasmid (pLSB-NAT) containing a Green Fluorescent Protein (GFP) gene for simple selection of E. coli transformants with sgRNA-containing plasmids. Homologous recombination (HR) templates were designed to carry the desired G4 motif mutations, which were introduced together with the pLSB-NAT into haploid cells through transformation and incorporated into the genome by homology-directed repair. Created in BioRender. Sabouri, N. (2025) https://BioRender.com/lweeenl ( B ) Genome browser track showing Cdc20 (catalytic subunit of DNA polymerase ϵ) ChIP-seq datasets (European Nucleotide Archive, accession number: PRJEB37862) performed in S. pombe . The Cdc20 enrichment profiles across adh1 + genomic region is shown, where the y -axis represents the normalized read density, reflecting the relative abundance of sequencing reads at each genomic position. The normalized read density values have been scaled to a range of 0 – 50 to facilitate visual comparison between the input and Cdc20 immunoprecipitant (IP) samples. The labels below the tracks show the Pombase forward and reverse strands; and the specific position of bioinformatically predicted G4s in chromosome III. The Adh1 G4 motif is denoted as G435 in the G4 track and identified using the algorithm (G ≥3 N 1–25 ) 3 G ≥3, where N can be 1 – 25 nucleotides .

Article Snippet: Finally, the eluates were purified using ChIP DNA Clean and Concentrate kit (ZYMO Research). qPCR was performed using primer pairs amplifying the upstream adh1 + G4 (SPCC13B11.01), lncRNA + (SPNCRNA.1156) or ade6 + (SPCC1322.13) (non-G4 control) regions.

Techniques: CRISPR, Cloning, Plasmid Preparation, Selection, Homologous Recombination, Transformation Assay, ChIP-sequencing, Sequencing, Comparison

Journal: Nucleic Acids Research

Article Title: CRISPR-Cas9 targeting of G-Quadruplex DNA in ADH1 promoter highlights its role in transcriptome and metabolome regulation

doi: 10.1093/nar/gkaf853

Figure Lengend Snippet: Functional analysis of Adh1 G4 motif in S. pombe . ( A ) Schematics showing the principles for the qPCR stop assay. Created in BioRender. Sabouri, N. (2025) https://BioRender.com/b29go06 ( B ) Genomic DNA from S. pombe strains harboring ADH1 WT or mutated (ADH1 5_G MUT or ADH1 4_G MUT) Adh1 G4 motifs were used as templates in qPCR stop assay performed in the absence of KCl or in the presence of KCl, with or without PhenDC3. The primer pairs were designed to flank the Adh1 G4, positive G4 control (lncRNA; SPNCRNA.1156) or non-G4 ( ade6 + ) sites. The graph shows the average values of three biological replicates. Error bars represent the standard deviation. * P < 0.05 and *** P < 0.001 according to two-sample t -test. ( C ) Schematics showing the principles for BG4-ChIP. Created in BioRender. Sabouri, N. (2025) https://BioRender.com/7k9322p ( D ) DNA from chromatin isolated from S. pombe strains harboring ADH1 G4 WT or mutated (ADH1 G4-TATA MUT or ADH1 G4 MUT) Adh1 G4 motifs were immunoprecipitated using BG4 antibody. The amount of immunoprecipitated DNA was analyzed by qPCR using primer pairs that flank the Adh1 G4, positive G4 control (lncRNA; SPNCRNA.1156) or non-G4 ( ade6 +) sites. Input C q value of each sample was used in normalization. The graph shows the average values of four experiments. Error bars represent the standard deviation. * P < 0.05 and *** P < 0.001 according to two-sample t -test. ( E ) Growth of S. pombe strains harboring ADH1 G4 WT or mutated (ADH1 G4-TATA MUT or ADH1 G4 MUT) Adh1 G4 motifs. The graph shows the average values of the doubling times of three experiments. Error bars represent the standard deviation. * P < 0.05 according to two-sample t -test.

Article Snippet: Finally, the eluates were purified using ChIP DNA Clean and Concentrate kit (ZYMO Research). qPCR was performed using primer pairs amplifying the upstream adh1 + G4 (SPCC13B11.01), lncRNA + (SPNCRNA.1156) or ade6 + (SPCC1322.13) (non-G4 control) regions.

Techniques: Functional Assay, Control, Standard Deviation, Isolation, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: CRISPR-Cas9 targeting of G-Quadruplex DNA in ADH1 promoter highlights its role in transcriptome and metabolome regulation

doi: 10.1093/nar/gkaf853

Figure Lengend Snippet: Mutation of Adh1 G4 affects adh1 levels and alters global cellular transcriptome ( A ) Realtime qPCR for adh1 + gene expression. Total RNA was isolated from S. pombe strains harboring ADH1 G4 WT or mutated (ADH1 G4-TATA MUT or ADH1 G4 MUT) Adh1 G4 motifs and reverse transcribed to cDNA using a combination of random hexamers and anchored oligo (dT). Primer pairs amplifying the adh1 + and ade6 + (non-G4) coding regions were used. act1 + and tdh1 + were used as reference genes for normalization. The graph shows the average values of three experiments. Error bars represent the standard deviation. ** P < 0.01 and *** P < 0.001 according to two-sample t -test. The Adh1 transcript levels in ADH1 G4-TATA MUT or ADH1 G4 MUT were reduced to ∼30% and ∼80% compared to ADH1 G4 WT, respectively. ( B ) H3K4me3 - ChIP to probe adh1 + locus. DNA from chromatin isolated from the three S. pombe strains were immunoprecipitated using H3K4me3 antibody. The amount of immunoprecipitated DNA was analyzed by qPCR using primer pairs that span promoter of adh1 sites. The top image shows the adh1 region examined and indicates the position of the primer pairs. Input C q value of each sample was used in normalization. The graph shows the average values of four experiments. Error bars represent the standard deviation. * P < 0.05 and ** P < 0.01 according to two-sample t -test. ( C ) PCA plot of the transcriptomic analyses to visualize the differences between three biological replicates of ADH1 G4 WT, ADH1 G4-TATA MUT, and ADH1 G4 MUT strains. The first principal component (PC 1) accounted for 40.08% and the second principal component (PC 2) accounted for 14.73% of the total variance in the dataset. Volcano plots showing significantly altered transcript levels between three comparison groups: ( D ) ADH1 G4-TATA MUT_vs_ ADH1 G4 WT, ( E ) ADH1 G4 MUT_vs_ ADH1 G4 WT, and ( F ) ADH1 G4-TATA MUT_vs_ ADH1 G4 MUT. Transcripts with significant changes in expression with false discovery rate (FDR) < 0.05 and log2 fold change > 0.1 are highlighted. Blue and pink dots show down- and upregulated DEGs, respectively. Eight important genes are marked that are associated to oxidoreduction process. GO pathway analyses bubble plots showing representative top significantly (FDR < 0.01) regulated pathways between three comparison groups: ( G ) ADH1 G4-TATA MUT_vs_ ADH1 G4 WT, ( H ) ADH1 G4 MUT_vs_ ADH1 G4 WT, and ( I ) ADH1 G4-TATA MUT_vs_ ADH1 G4 MUT. ( J ) Venn diagram showing common significantly altered genes between the comparison groups associated with oxidoreduction pathway. ( K ) Heatmap profile showing expression (log 2 (counts per million), scaled by genes) of eight genes in oxidoreduction pathways that are significantly altered as well as commonly found between three comparison groups.

Article Snippet: Finally, the eluates were purified using ChIP DNA Clean and Concentrate kit (ZYMO Research). qPCR was performed using primer pairs amplifying the upstream adh1 + G4 (SPCC13B11.01), lncRNA + (SPNCRNA.1156) or ade6 + (SPCC1322.13) (non-G4 control) regions.

Techniques: Mutagenesis, Gene Expression, Isolation, Reverse Transcription, Standard Deviation, Immunoprecipitation, Comparison, Expressing