bisulfite sequencing methods  (Statham Instruments Inc)

Journal: Nucleic Acids Research

Article Title: The splicing regulator SLU7 is required to preserve DNMT1 protein stability and DNA methylation

doi: 10.1093/nar/gkab649

Figure Lengend Snippet: SLU7 knockdown compromises DNA methylation. ( A ) SLU7 knockdown induces a decrease in 5-methyl-cytosine (5-mC) content. DNA methylation levels were analyzed by ELISA 72 h after transfection with siSLU7 or control siGL in the human hepatoma cell lines PLC/PRF/5 and HepG2, the colorectal carcinoma cell line HCT116 and the lung cancer cell line H358. The percentage of 5-mC content is indicated. * P < 0.05, ** P < 0.01, *** P < 0.001. Western blot analysis confirms SLU7 knockdown. ACTIN was used as loading control. At least three independent experiments in duplicate were performed for each cell line. ( B ) Representative images of 5-methyl cytosine (5-mC) immunodetection (in green) in PLC/PRF/5 (top) and HepG2 (bottom) cells 72 h after transfection with siSLU7 or control siGL. Nuclei were stained with DAPI (in blue). Scale bar: 10 μm. ( C , D ) Scatter plots of reduced representation bisulfite sequencing (RRBS) DNA methylation β-values of siGL versus siDNMT1 (C) or siGL versus siSLU7 (D) PLC/PRF/5 cells 72 h after transfection. Fold change methylation differences higher than 0.6 were highlighted within a green triangle (hypomethylated) or a red triangle (hypermethylated). Graphs represent the percentage of hypomethylated (FC < 0,6) and hypermethylated (FC > 0,6) CpGs. ( E ) Number of single CpGs, genes with CpGs and CpGs within CpG islands (CGIs) found hypomethylated (FC < 0.6) in PLC/PRF/5 cells 72 h after transfection with siDNMT1 (left) or siSLU7 (right) compared to control siGL cells. ( F) Venn diagram of the overlap between genes (top) or CpGs (bottom) found hypomethylated (FC < 0,6) in siSLU7 and siDNMT1 PLC/PRF/5 transfected cells when compared with control siGL cells.

Article Snippet: Genome-wide DNA methylation analysis was carried out using the reduced representation bisulfite sequencing method ( ) by Active Motif (Carlsbad, CA, USA).

Techniques: Knockdown, DNA Methylation Assay, Enzyme-linked Immunosorbent Assay, Transfection, Control, Western Blot, Immunodetection, Staining, Methylation Sequencing, Methylation

Journal: Nucleic Acids Research

Article Title: The splicing regulator SLU7 is required to preserve DNMT1 protein stability and DNA methylation

doi: 10.1093/nar/gkab649

Figure Lengend Snippet: SLU7 knockdown activates transcription of genes silenced by DNA methylation. ( A) Graphs reporting DNA methylation levels (β-values obtained by Reduced Representation Bisulfite Sequencing (RRBS) analysis) of TRIM47 , TFF3 and DLG3 gene regions found hypomethylated in both SLU7 (siSLU7, in red) and DNMT1 (siDNMT1, in green) knockdown PLC/PRF/5 cells compared to control siGL (in blue) cells 72 h after transfection. ( B ) Representative methylation-specific PCR (MSP) results for DNA methylation analysis of TRIM47 , TFF3 and DLG3 regions shown in (A). U: unmethylated and M: methylated. (C) Expression by RT-qPCR of TRIM47 and DLG3 in PLC/PRF/5 cells 72 h after transfection with siSLU7 or control siGL. ( D , E ) DNA methylation analysis by MSP of RASSF1A and GNMT promoter regions (D) and RASSF1A and GNMT expression by RT-qPCR (E) in PLC/PRF/5 cells 72 h after transfection with siSLU7, siDNMT1 or control siGL. U: unmethylated and M: methylated. ( F , G ) DNA methylation analysis by MSP of GNMT promoter region (F) and GNMT expression by RT-qPCR (G) in HepG2 and HCT-116 cells 72 h after transfection with siSLU7, siDNMT1 or control siGL. U: unmethylated and M: methylated. ( H ) Expression by RT-qPCR of cancer testis antigens ( MAGE-A1 and MAGE-A7 ), endogenous retroviruses ( envFc2) and imprinted genes ( H19) in PLC/PRF/5, HepG2, HCT116 and H358 cells 72 h after transfection with siSLU7 or control siGL. RPLP0 expression was used as housekeeping gene in (C, E, G, H). * P < 0.05, ** P < 0.01, *** P < 0.001. All experiments were performed at least three times with biological duplicates per condition.

Article Snippet: Genome-wide DNA methylation analysis was carried out using the reduced representation bisulfite sequencing method ( ) by Active Motif (Carlsbad, CA, USA).

Techniques: Knockdown, DNA Methylation Assay, Methylation Sequencing, Control, Transfection, Methylation, Expressing, Quantitative RT-PCR

Journal: Neuro-Oncology

Article Title: MGMT promoter methylation level in newly diagnosed low-grade glioma is a predictor of hypermutation at recurrence

doi: 10.1093/neuonc/noaa059

Figure Lengend Snippet: Validation of bisulfite amplicon sequencing (BSAS) method for assaying MGMT promoter methylation level. (A) Association between methylation level at CpG site +174 bp from TSS assayed by BSAS and by Infinium Array across samples. (B) Association between methylation level at each CpG site assayed by BSAS and MGMT mRNA level across samples. Pearson correlations and P-values adjusted for multiple hypothesis testing are shown for each CpG site labeled by distance from TSS. Sequence annotations are shown for regions assayed by methylation-specific PCR (MSP), an enhancer, and regions for which associations between methylation and transcription have previously been shown (hotspot, DMR, concordant region).

Article Snippet: Comparison of methylation measures at this particular CpG site from both methods revealed a high degree of concordance across samples ( ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 2 caption a7 Validation of bisulfite amplicon sequencing (BSAS) method for assaying MGMT promoter methylation level. (A) Association between methylation level at CpG site +174 bp from TSS assayed by BSAS and by Infinium Array across samples. (B) Association between methylation level at each CpG site assayed by BSAS and MGMT mRNA level across samples.

Techniques: Biomarker Discovery, Amplification, Sequencing, Methylation, Labeling