Journal: bioRxiv

Article Title: Commercial ChIP-Seq library preparation kits performed differently for different classes of protein targets

doi: 10.1101/2022.04.19.488832

Figure Lengend Snippet: Experimental design. Diagram outlining the experimental design for comparing the Bioo NEXTflex, Diagenode MicroPlex, Roche KAPA HyperPrep, and New England BioLabs NEBNext Ultra II ChIP-Seq library preparation protocols. Two independent ChIP-Seq experiments (sets 1 and 2) were carried out to assess H3K4me3 and H3K27me3 libraries, and three independent ChIP-Seq experiments (sets 1-3) were performed to evaluate CTCF libraries. For H3K4me3 and H3K27me3, six different amounts of input DNA (10, 5, and 1 ng and 500, 250, and 100 pg), were used for library construction; however, due to low recovery of CTCF ChIP DNA, only a single DNA input was used for library construction. The number of amplification cycles for each protocol followed the manufacturers’ recommendations based on the amount of input DNA.

Article Snippet: ChIP libraries were prepared using the NEXTflex ChIP-Seq Kit (Bioo Scientific, now PerkinElmer) following the manufacturer’s protocol.

Techniques: ChIP-sequencing, Amplification

Journal: bioRxiv

Article Title: Commercial ChIP-Seq library preparation kits performed differently for different classes of protein targets

doi: 10.1101/2022.04.19.488832

Figure Lengend Snippet: Performance of protocols for CTCF ChIP-Seq. Libraries for CTCF sets 1-3 are presented. A. Number of peaks called in each library. B. Percentage of peaks with CTCF motifs within 100 bp of peak summits. C. Histogram of distance between peak summit and CTCF motif.

Article Snippet: ChIP libraries were prepared using the NEXTflex ChIP-Seq Kit (Bioo Scientific, now PerkinElmer) following the manufacturer’s protocol.

Techniques: ChIP-sequencing

Journal: Molecular Neurobiology

Article Title: Gcm1 Orchestrates Lef1 Expression in Folate Deficiency-Induced Neural Tube Defects

doi: 10.1007/s12035-025-05346-x

Figure Lengend Snippet: H4 acetylation enrichment at the Gcm1 promoter in mESC without folate supplementation. A Volcano plot of expression profile chip in mESCs for six generations with (+) or without folate supplementation (−). Upregulated genes are shown in red, downregulated genes are shown in blue. B Functional classification of significantly enriched GO terms in mESCs with (+) or without folate supplementation (−). X-axis: specific GO terms from biological processes, cellular components, and molecular functions. Right Y-axis: number of differentially expressed genes (DEG) in background genes. Left Y-axis: percentage of genes per term relative to the total number of genes annotated across all displayed GO terms (scale: 0–100%). C The mRNA expression of Gcm1 in mESCs with (+) or without folate supplementation (−) for six generations ( n = 3). The P values were calculated with an unpaired t test. *** P < 0.001. D Increased expression of Gcm1 in mESCs without folate supplementation. Top panel: western blot analysis of Gcm1 in C57BL/6 mESC with (+) or without folate supplementation (−) for six generations; quantification of the western blot signal intensities is shown in the down panel ( n = 3). The P values were calculated with an unpaired t test. * P < 0.05. E ChIP-qPCR analysis of H4-Ac enrichment in the Gcm1 promoter of mESC with (+) or without folate supplementation (−) for six generations ( n = 3). The P values were calculated with one-way ANOVA plus post-hoc test. ** P < 0.01; ns denotes no significance. F The mRNA expressions of Gcm1 and the neural ectoderm markers Nestin and Sox2 in mESC with (+) or without folate supplementation (−) and the 10X FA group ( n = 3). Folate (+) denotes mESC were cultured in medium with folate supplementation for six generations. Folate (−) denotes mESC were cultured in medium without folate supplementation for six generations. 10 X FA denotes 40 mg/L folate-treated mESC. The P values were calculated with one-way ANOVA plus post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001. G The enrichment of H4-ac in the Gcm1 promoter region in mESC with (+) or without folate supplementation (−) and the 10X FA group ( n = 3). 10 X FA denotes 40 mg/L folate-treated mESC. The P values were calculated with one-way ANOVA plus post-hoc test. *** P < 0.001; ns denotes no significance

Article Snippet: Gcm1 ChIP-seq libraries were prepared using the KAPA HTP Library Preparation Kit complemented with NEXTflex DNA Barcodes from Bioo Scientific and performed by Novogene Corporation (Beijing, China).

Techniques: Expressing, Functional Assay, Western Blot, ChIP-qPCR, Cell Culture

Journal: Molecular Neurobiology

Article Title: Gcm1 Orchestrates Lef1 Expression in Folate Deficiency-Induced Neural Tube Defects

doi: 10.1007/s12035-025-05346-x

Figure Lengend Snippet: CBP mediates the acetylation of Gcm1 in mESC without folate supplementation. A Co-IP assay identified a strong interaction between Gcm1 and acetylation in mESC with ( +) or without folate supplementation ( −), using anti-Gcm1 for IP and anti-Ac for IB. Histone H3 was used as a protein loading control ( n = 3). B Co-IP assay identified the interaction between Gcm1 and CBP in mESC with ( +) or without folate supplementation ( −), using anti-Gcm1 for IP and anti-CBP for IB. Histone H3 was used as a protein loading control ( n = 3). C Co-IP assay with CBP knockdown identified a reduced interaction between Gcm1 and CBP in mESC with ( +) or without folate supplementation ( −). CBP was knocked down by adenovirus, followed by culturing in medium without folate supplementation for six generations. All adenoviral infection was performed at 100 MOI for 48 h. IP was performed using an anti-Gcm1 antibody, IB using antibody anti-CBP. Histone H3 was used as a protein loading control ( n = 3). D Co-IP assay identified a reduced interaction between Gcm1 and acetylation in mESC without folate supplementation with CBP-30 inhibitor, using anti-Gcm1 for IP and anti-Ac for IB. Histone H3 was used as a protein loading control ( n = 3). E Co-IP assay identified effects of CBP-30 in mESC with ( +) or without folate supplementation ( −). CBP-30 treatment led to a significant decrease in CBP, Gcm1, and H4-Ac levels, as well as reduced Gcm1 and H4-Ac binding; IP was performed using an anti-Gcm1 antibody, and IB using anti-CBP and anti-Ac antibodies. Histone H3 was used as a protein loading control ( n = 3)

Article Snippet: Gcm1 ChIP-seq libraries were prepared using the KAPA HTP Library Preparation Kit complemented with NEXTflex DNA Barcodes from Bioo Scientific and performed by Novogene Corporation (Beijing, China).

Techniques: Co-Immunoprecipitation Assay, Control, Knockdown, Infection, Binding Assay

Journal: Molecular Neurobiology

Article Title: Gcm1 Orchestrates Lef1 Expression in Folate Deficiency-Induced Neural Tube Defects

doi: 10.1007/s12035-025-05346-x

Figure Lengend Snippet: Gcm1 regulates the Wnt signaling pathway in mESC without folate supplementation. A The top 10 enriched pathways of NTDs candidate genes were analyzed by KEGG. The X-axis represents the log10 value of the binomial raw P value. B The top 10 enriched pathways of NTDs candidate genes were analyzed by PANTHER. The X-axis represents the log10 value of the binomial raw P value. C Gcm1 reads density signals distribution in GeneBody in mESC with (blue) or without folate supplementation (red). Profiles show binding distribution across gene regions: − 2 kb upstream of transcription start sites, gene bodies, and + 2 kb downstream of transcription end sites. D Gene Ontology of genes associated with different Gcm1 ChIP-peaks in mESCs with or without folate supplementation (the top 15 categories are shown). The X-axis represents the log10 value of the binomial raw P value. E Venn diagrams showing overlapping genes among Gcm1-enriched genes from ChIP-seq in ESC without folate supplementation, which validate genes in human NTDs in fetal brain with NanoString and NTD-associated genes from GeneCards. The overlap of the Venn diagram is determined based on the target gene. F Bubble plot showing KEGG pathways of overlapping genes among Gcm1-ChIP-seq with or without folate supplementation, expression profile chip, and NTD-associated genes. The term “Gene Ratio” refers to the ratio of the number of genes enriched in a certain GO entry to the total number of input genes during enrichment analysis

Article Snippet: Gcm1 ChIP-seq libraries were prepared using the KAPA HTP Library Preparation Kit complemented with NEXTflex DNA Barcodes from Bioo Scientific and performed by Novogene Corporation (Beijing, China).

Techniques: Binding Assay, ChIP-sequencing, Expressing

Journal: Molecular Neurobiology

Article Title: Gcm1 Orchestrates Lef1 Expression in Folate Deficiency-Induced Neural Tube Defects

doi: 10.1007/s12035-025-05346-x

Figure Lengend Snippet: Gcm1 regulates Lef1 in mESC without folate supplementation. A mRNA levels of Lef1 in mESC with ( +) or without folate supplementation ( −), as determined by RT-qPCR and GAPDH which were used as loading controls ( n = 3). The P values were calculated with an unpaired t test. ** P < 0.01. B IGV visualization showing the differential Gcm1 peaks in Lef1 promoter in mESC with ( +) or without folate supplementation ( −) by MACS2 analysis. The blank rectangle denotes promoter position in Lef1,; chr3 denotes chromatin 3. C The ChIP-qPCR assay shows Gcm1 could bind to Lef1 promoter in mESC without folate supplementation ( n = 3). The P values were calculated with an unpaired t test. * P < 0.05, ** P < 0.01. D Lef1 and Gcm1 levels were measured via western blotting in mESC without folate supplementation after Gcm1 knockdown with siRNA. mESC were transfected with si-Gcm1 for 48 h and were then collected for analysis. Histone H3 was used as loading controls. Quantification of the western blot signal intensities was shown in the right panel ( n = 3). The P values were calculated with an unpaired t test. ** P < 0.01, *** P < 0.001. E mRNA levels of Lef1 in Gcm1 overexpression in mESC with folate supplementation, as determined by RT-qPCR and GAPDH which were used as loading controls ( n = 3). OE denotes Gcm1 overexpression. The P values were calculated with an unpaired t test. *** P < 0.001. F Lef1 and Gcm1 levels were measured via western blotting in mESC with folate supplementation after Gcm1 overexpression for 72 h. mESC were transfected with Gcm1 plasmid for 72 h and were then collected for analysis. H3 was used as loading controls. Quantification of the western blot signal intensities was shown in the right panel ( n = 3). OE denotes Gcm1 overexpression. The P values were calculated with an unpaired t test. * P < 0.05, ** P < 0.01. G The ChIP-qPCR assay shows the binding of β- catenin in different positions of the Gcm1 promoter with ( +) or without folate supplementation ( −) ( n = 3). TBE, TCF binding element; TSS, transcription start sites. The P values were calculated with an unpaired t test. * P < 0.05, ns denotes no significance. H Schematic representation of the genome structure of human GCM1 and mouse Gcm1. Ex is represented for exon; Int is represented for intron. I Schematic representation of the luc- Int2-TBE7-mut reporter constructs for reporter assays (left panel). Luciferase reporter gene assays showed that mutations within the TBE7 and its mutant in response to β -catenin signaling were evaluated by luciferase assays in HEK 293 T (right panel) ( n = 3). The P values were calculated with an unpaired t test.* P < 0.05

Article Snippet: Gcm1 ChIP-seq libraries were prepared using the KAPA HTP Library Preparation Kit complemented with NEXTflex DNA Barcodes from Bioo Scientific and performed by Novogene Corporation (Beijing, China).

Techniques: Quantitative RT-PCR, ChIP-qPCR, Western Blot, Knockdown, Transfection, Over Expression, Plasmid Preparation, Binding Assay, Construct, Luciferase, Mutagenesis

Journal: Molecular Neurobiology

Article Title: Gcm1 Orchestrates Lef1 Expression in Folate Deficiency-Induced Neural Tube Defects

doi: 10.1007/s12035-025-05346-x

Figure Lengend Snippet: Gcm1 and Lef1 are upregulated in NTDs mice under folate deficiency. A Morphology of CD-1 mouse embryos with low-folate diet induced from E9.5; left panel is the control mouse fetus, and right panel is the anencephalous and spina bifida. The arrowhead is where the NTDs located. Scale bar, 1 mm. B Lef1 and Gcm1 levels were measured via western blotting in E9.5d NTDs mouse embryos. Quantitative analysis of the signal intensities was shown in down panel ( n = 3). The P values were calculated with unpaired t test. * P < 0.05, ** P < 0.01. C : Immunohistochemical analysis of Gcm1 and Lef1 in the brain of NTDs mouse at E9.5 (left panel). Quantitative analysis of the signal intensity was shown in the right panel ( n = 3). Scale bars, 100 μm. **** P < 0.0001. The P values were calculated with Wilcoxon Mann–Whitney test, **** P < 0.0001. D Immunofluorescence staining results of Gcm1 and Lef1 proteins in E9.5 NTD mouse embryo brain and quantitative analysis of the corresponding fluorescence signal intensity were shown in the right panel ( n = 3). The P values were calculated with Wilcoxon Mann– Whitney test; **** P < 0.0001

Article Snippet: Gcm1 ChIP-seq libraries were prepared using the KAPA HTP Library Preparation Kit complemented with NEXTflex DNA Barcodes from Bioo Scientific and performed by Novogene Corporation (Beijing, China).

Techniques: Control, Western Blot, Immunohistochemical staining, MANN-WHITNEY, Immunofluorescence, Staining, Fluorescence

Journal: Molecular Neurobiology

Article Title: Gcm1 Orchestrates Lef1 Expression in Folate Deficiency-Induced Neural Tube Defects

doi: 10.1007/s12035-025-05346-x

Figure Lengend Snippet: Gcm1 and Lef1 are correlated in low folate human NTDs. A Folate concentrations in control fetal brain tissue and NTDs fetal brain tissue ( n = 12). The P values were calculated with unpaired t test. * P < 0.05. B The expression of Gcm1 and Lef1 in control fetal brain tissue and NTDs human fetal brain tissue, as determined by NanoString ( n = 12). The P values were calculated with Mann–Whitney U test. * P < 0.05, *** P < 0.001. C Correlation analysis of Gcm1 expression and Lef1 mRNA expression in low folate NTDs fetal brain tissues ( n = 12). D The expression of Gcm1 in control and low folate NTDs fetal brain, as determined by western blot ( n = 12). Histone H3 was used as loading controls. The P values were calculated with unpaired t test. E The expression of Lef1 in control and low folate NTDs fetal brain, as determined by western blot. NTD samples (N) were matched with controls (C) by gestational age with paired samples. Histone H3 was used as loading controls. Quantification of the western blot signal intensities are showing in the right panel ( n = 6). The P values were calculated with unpaired t test. * P < 0.05

Article Snippet: Gcm1 ChIP-seq libraries were prepared using the KAPA HTP Library Preparation Kit complemented with NEXTflex DNA Barcodes from Bioo Scientific and performed by Novogene Corporation (Beijing, China).

Techniques: Control, Expressing, MANN-WHITNEY, Western Blot