Journal: Chemical Science

Article Title: Diagnostics and analysis of SARS-CoV-2: current status, recent advances, challenges and perspectives

doi: 10.1039/d2sc06665c

Figure Lengend Snippet: Examples of commercial molecular diagnostic assays for the detection of SARS-CoV-2 a

Article Snippet: Agena Bioscience, Inc. , MassARRAY SARS-CoV-2 Panel , rRT-PCR, MALDI-TOF , N gene (N1, N2, and N3 regions), ORF1, and ORF1ab , Nasopharyngeal swabs, oropharyngeal swabs, nasal and mid-turbinate swabs , 310–2500 copies per mL , 98.9% , 100% , H , .

Techniques: Diagnostic Assay, Amplification, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Microarray, Hybridization, Kinetic Assay, Helicase-dependent Amplification

Journal: Chemical Science

Article Title: Diagnostics and analysis of SARS-CoV-2: current status, recent advances, challenges and perspectives

doi: 10.1039/d2sc06665c

Figure Lengend Snippet: Comparison of the three types of SARS-CoV-2 antibody assay diagnostic kits

Article Snippet: Agena Bioscience, Inc. , MassARRAY SARS-CoV-2 Panel , rRT-PCR, MALDI-TOF , N gene (N1, N2, and N3 regions), ORF1, and ORF1ab , Nasopharyngeal swabs, oropharyngeal swabs, nasal and mid-turbinate swabs , 310–2500 copies per mL , 98.9% , 100% , H , .

Techniques: Comparison, Diagnostic Assay, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, High Throughput Screening Assay

Journal: Chemical Science

Article Title: Diagnostics and analysis of SARS-CoV-2: current status, recent advances, challenges and perspectives

doi: 10.1039/d2sc06665c

Figure Lengend Snippet: Types of SARS-CoV-2 pseudovirus neutralizing tests

Article Snippet: Agena Bioscience, Inc. , MassARRAY SARS-CoV-2 Panel , rRT-PCR, MALDI-TOF , N gene (N1, N2, and N3 regions), ORF1, and ORF1ab , Nasopharyngeal swabs, oropharyngeal swabs, nasal and mid-turbinate swabs , 310–2500 copies per mL , 98.9% , 100% , H , .

Techniques: Plasmid Preparation, Expressing, Virus, Luciferase, Mutagenesis, Clinical Proteomics, Bioprocessing

Journal: Cancer Communications

Article Title: Intranuclear paraspeckle‐circular RNA TACC3 assembly forms RNA‐DNA hybrids to facilitate MASH‐related hepatocellular carcinoma growth in an m 6 A‐dependent manner

doi: 10.1002/cac2.70061

Figure Lengend Snippet: m 6 A‐modified circTACC3 is up‐regulated in MASH‐related HCC. (A) Schematic representation of m 6 A‐circRNA epitranscriptomic microarray assay. (B) Hierarchical clustering heatmap of differentially m 6 A modified circRNAs from MASH‐related HCC tumor and paired peritumoral normal tissues (m 6 A‐circRNA epitranscriptomic microarray assay; absolute m 6 A modification quantity shown, n = 5). (C) Venn diagram of overlapping circRNAs in MASH‐related HCC tumors with concurrent increases in absolute m 6 A modification quantity, relative m 6 A modification rate, and relative expression levels. (D‐E) The circTACC3 level in MASLD tissues, MASH‐related HCC tumor tissues, and paired peritumoral normal tissues ( n = 62) determined by ISH assay (D) and corresponding expression score analysis (E). ** P < 0.01; *** P < 0.001; NS , not significant. (F) MeRIP assay shows the enrichment of m 6 A‐modified circTACC3 in MASLD tissues, MASH‐related HCC tumor tissues and paired peritumoral normal tissues ( n = 3). (G) 3D‐FISH performed on MASH‐related HCC tumor and paired peritumoral normal tissue derived organoids. Left upper panel shows circTACC3 (red) and DAPI (blue). Left lower panel shows the merge in 3D view. Right upper panel shows representative z‐stack layer capture. Right lower panel shows depth coding. (H) Nuclear‐cytoplasmic fractionation assay determined circTACC3 expression in nuclear and cytoplasmic fractionation, respectively ( n = 4). ** P < 0.01; *** P < 0.001; NS , not significant. (I) Schematic representation of exon 4 back‐splicing, circTACC3 forming, and the design of indicated primers. (J) Electrophoresis of RT‐PCR product amplified from cDNA or gDNA. (K) The expression of circTACC3 and TACC3 homologous mRNA from RNA with or without RNase R treatment. (L) Levels of circTACC3 and TACC3 mRNA in indicated cells that were treated with or without actinomycin D ( n = 4). *** P < 0.001. Abbreviations: m 6 A, N6‐methyladenosine; MASLD, metabolic dysfunction‐associated steatotic liver disease; MASH, metabolic dysfunction‐associated steatohepatitis; HCC, hepatocellular carcinoma; T, tumor tissues; PT, peritumoral normal tissue; ISH, in situ hybridization‐ immunofluorescence; MeRIP, methylated RNA immunoprecipitation; 3D, three dimensions; FISH, fluorescence in situ hybridization; NAS, non‐alcoholic fatty liver disease activity score; RT‐PCR, reverse transcription‐PCR.

Article Snippet: Immunocomplexes were incubated with the fluorescein‐conjugated secondary antibody (# C61012 ‐05, mouse, Li‐Cor, NE, US; # C80416 ‐08, rabbit, Li‐Cor) and then detected using the Odyssey fluorescence scanner (Li‐Cor).

Techniques: Modification, Microarray, Expressing, In Situ Hybridization, Derivative Assay, Fractionation, Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Amplification, Immunofluorescence, Methylation, RNA Immunoprecipitation, Fluorescence, Activity Assay, Reverse Transcription

Journal: Cancer Communications

Article Title: Intranuclear paraspeckle‐circular RNA TACC3 assembly forms RNA‐DNA hybrids to facilitate MASH‐related hepatocellular carcinoma growth in an m 6 A‐dependent manner

doi: 10.1002/cac2.70061

Figure Lengend Snippet: circTACC3 m 6 A modification is associated with its intracellular localization. (A) Representative image of H&E staining of MASH‐related HCC tumor and paired peritumoral normal tissue derived organoids with relatively low (#1) and high (#2) circTACC3 expression, treated with or without PA + OA. (B) 3D fluorescence scanning of 3‐OH BrDU probe (green), anti‐Ki‐67 antibody (magenta), and DAPI (blue) indicated tissue derived organoids, treated with or without PA + OA; maximum projection of signals is shown. (C‐D) Assays of fluorescence staining with the 3‐OH BrDU probe were conducted on the cell lines with different nuclear circTACC3 level (C, n = 6) and circTACC3 knockout cell strains or negative controls (D, n = 9). The numbers on the Y‐axis represent the apoptotic rate normalized to baseline of 1. ** P < 0.01; *** P < 0.001; NS , not significant. (E) MeRIP assay shows an enrichment of m 6 A‐modified circTACC3 in cytoplasmic and nuclear fraction of HCCLM3 cells. (F) Representation of predicted m 6 A modification motif of circTACC3 (predicted using the SRAMP website). (G) Absolute quantitative RT‐qPCR of m 6 A RNA shows m 6 A modification ratio of intra‐nuclear circTACC3 in the individual motif scale. (H) The m 6 A modification levels of circTACC3 were evaluated by MeRIP in m 6 A inhibitor treated group compared to control (DMSO) group, and in PA + OA treated group compared to control (Mock) group, respectively ( n = 3). *** P < 0.001. (I) Nucleo‐plasmic fractionation to evaluate circTACC3 expression after indicated treatment ( n = 4). *** P < 0.001. (J) 3D‐FISH conducted on MASH‐related HCC tumor tissue derived organoids demonstrates the 3D distribution of circTACC3 (red) in nuclei (blue). (K) H&E staining of MASH‐related HCC tumor tissue derived organoids treated with PA + OA and/or m 6 A intervention. Abbreviations: PA, palmitic acid; OA, oleic acid; T, tumor tissues; PT, peritumoral normal tissue; H&E, hematoxylin and eosin; MeRIP, methylated RNA immunoprecipitation; m 6 A, N6‐methyladenosine; NAS, Non‐alcoholic fatty liver disease activity score; SAH, S‐adenosylhomocysteine; DAA, 3‐deazaadenosine; FISH, fluorescence i n situ hybridization.

Article Snippet: Immunocomplexes were incubated with the fluorescein‐conjugated secondary antibody (# C61012 ‐05, mouse, Li‐Cor, NE, US; # C80416 ‐08, rabbit, Li‐Cor) and then detected using the Odyssey fluorescence scanner (Li‐Cor).

Techniques: Modification, Staining, Derivative Assay, Expressing, Fluorescence, Knock-Out, Quantitative RT-PCR, Control, Fractionation, Methylation, RNA Immunoprecipitation, Activity Assay, Hybridization

Journal: Cancer Communications

Article Title: Intranuclear paraspeckle‐circular RNA TACC3 assembly forms RNA‐DNA hybrids to facilitate MASH‐related hepatocellular carcinoma growth in an m 6 A‐dependent manner

doi: 10.1002/cac2.70061

Figure Lengend Snippet: The m 6 A modification of circTACC3 regulates its interaction with NONO/p54 nrb . (A) Western blot validation of NONO/p54 nrb pulldown by F2‐circTACC3 in HCCLM3 cells. (B) RIP assay shows enrichment of circTACC3 in NONO/p54 nrb ‐associated RNA in HCCLM3 cells ( n = 4). *** P < 0.001. (C) FLIM‐FRET assay (left panel) and the schematic diagram of FLIM‐FRET (right panel) in PA and OA treated HCCLM3 and HepG2 cells. (D) CLIP assay followed by RT‐PCR gel electrophoresis in HCCLM3 cells. (E) The location of circTACC3 (red), NONO/p54 nrb (green), and LncNEAT1 (magenta) in nuclei (blue) was evaluated in HCCLM3 and HepG2 cells following PA + OA treatment. (F) The distribution of circTACC3 (red) in nuclei (blue) was assessed following NONO/p54 nrb or LncNEAT1 interference in PA and OA treated HCCLM3 and HepG2 cells, respectively. (G) Nucleo‐plasmic fractionation shows the altered intracellular localization of circTACC3 following NONO/p54 nrb and LncNEAT1 interference in PA and OA treated HCCLM3 and HepG2 cells ( n = 4). *** P < 0.001. (H) RIP assay shows the enrichment of circTACC3 in NONO/p54 nrb ‐associated RNA following indicated treatment ( n = 3) in HCCLM3 and HepG2 cells. * P < 0.05; ** P < 0.01; *** P < 0.001; NS , not significant. (I‐K) Representative fluorescence images show the distribution of circTACC3 (red) and NONO/p54 nrb (green) in nuclei (blue) in HCCLM3, HepG2 cells, and Hep3B cells expressed ectopic circTACC3 following indicated treatment. (L) RIP assay shows enrichment of wildtype/m 6 A modification site mutant circTACC3 in NONO/p54 nrb ‐associated RNA in PA and OA treated HCCLM3 and HepG2 cells. Data normalization by dividing anti‐NONO/p54 nrb RIP and IgG RIP values by their respective Input group data ( n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001; NS , not significant. Abbreviations: NONO/p54 nrb , non‐POU domain‐containing octamer‐binding protein; CLIP, ultraviolet cross‐linking immunoprecipitation; RT‐PCR, reverse transcription‐PCR; FLIM‐FRET, fluorescence lifetime imaging microscopy‐forster resonance energy transfer; MFD, Minimal Fraction of Donor; PA, palmitic acid; OA, oleic acid; T, tumor tissues; PT, peritumoral normal tissue; NAS, non‐alcoholic fatty liver disease activity score; SAH, S‐adenosylhomocysteine; DAA, 3‐deazaadenosine; Mut, mutation; RIP, RNA immunoprecipitation.

Article Snippet: Immunocomplexes were incubated with the fluorescein‐conjugated secondary antibody (# C61012 ‐05, mouse, Li‐Cor, NE, US; # C80416 ‐08, rabbit, Li‐Cor) and then detected using the Odyssey fluorescence scanner (Li‐Cor).

Techniques: Modification, Western Blot, Biomarker Discovery, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Fractionation, Fluorescence, Mutagenesis, Binding Assay, Cross-linking Immunoprecipitation, Reverse Transcription, Imaging, Microscopy, Förster Resonance Energy Transfer, Activity Assay, RNA Immunoprecipitation

Journal: Cancer Communications

Article Title: Intranuclear paraspeckle‐circular RNA TACC3 assembly forms RNA‐DNA hybrids to facilitate MASH‐related hepatocellular carcinoma growth in an m 6 A‐dependent manner

doi: 10.1002/cac2.70061

Figure Lengend Snippet: circTACC3‐R loop structure formation in the MASH‐related HCC genome. (A) Schematic representation of procedure to identify circR loops and circTACC3‐R loops. (B) Dot‐blot assay to validate the R loop structure. S9.6 antibody was used to specifically recognize DNA‐RNA hybrids. (C) DRIP assay to identify circTACC3 enrichment in R loop structures in HCCLM3 cells ( n = 4). ** P < 0.01; *** P < 0.001; ND , not detection. (D) Representative fluorescence images show the expression and aggregation of circTACC3 and R loops as glow and spectrum signal intensities in MASLD tissues, MASH‐related HCC tumor tissues and paired peritumoral normal tissues, respectively. (E) The 3D distribution of circTACC3 (red) and R loops (green) were showed by 3D‐FISH‐IF in tissue derived organoids. (F‐I) Representative fluorescence images (F, H) and peak graphs of the linear ROI (region of interest) (G, I) show the location of the indicated molecules in HCC cells following PA + OA treatment. The linear ROI is represented by a solid line in the fluorescence graph. (J) The localization of circTACC3 (red) and S9.6‐stained R loops (green) were validated after NONO/p54 nrb or LncNEAT1 interference in PA and OA treated HCCLM3 and HepG2 cells, respectively. Abbreviations: PA, palmitic acid; OA, oleic acid; DRIP, DNA‐RNA immunoprecipitation; IF, immunofluorescence; FISH, fluorescence i n situ hybridization; MASLD, metabolic dysfunction‐associated steatotic liver disease; MASH, metabolic dysfunction‐associated steatohepatitis; HCC, hepatocellular carcinoma; T, tumor tissues; PT, peritumoral normal tissue; NAS, non‐alcoholic fatty liver disease activity score; NONO/p54 nrb , non‐POU domain‐containing octamer‐binding protein; SAH, S‐adenosylhomocysteine; DAA, 3‐deazaadenosine; Mut, mutation; ROI, region of interest.

Article Snippet: Immunocomplexes were incubated with the fluorescein‐conjugated secondary antibody (# C61012 ‐05, mouse, Li‐Cor, NE, US; # C80416 ‐08, rabbit, Li‐Cor) and then detected using the Odyssey fluorescence scanner (Li‐Cor).

Techniques: Dot Blot, Fluorescence, Expressing, Derivative Assay, Staining, RNA Immunoprecipitation, Immunofluorescence, Hybridization, Activity Assay, Binding Assay, Mutagenesis

Journal: Cancer Communications

Article Title: Intranuclear paraspeckle‐circular RNA TACC3 assembly forms RNA‐DNA hybrids to facilitate MASH‐related hepatocellular carcinoma growth in an m 6 A‐dependent manner

doi: 10.1002/cac2.70061

Figure Lengend Snippet: circTACC3‐R loop formation is regulated by lipid overload and m 6 A modification. (A) 3D‐distribution of circTACC3 (glow) and S9.6‐stained R loops (spectrum) in nuclei (blue) of MASH‐HCC tissue derived organoids after indicated treatment. (B‐C) DRIP assay shows the enrichment of circTACC3 in R loop structure following m 6 A modification interference in PA and OA treated HCCLM3 and HepG2 cells ( n = 3). * P < 0.05; ** P < 0.01. (D) Representative fluorescence images show S9.6‐stained R loops (green) in PA + OA induced HCC cells transfected with the RNase H1‐Tet‐On system after treatment with or without Dox. (E) Nucleo‐plasmic fractionation shows the altered intracellular localization of circTACC3 in PA and OA treated HCCLM3 and HepG2 cells ( n = 4). *** P < 0.001. (F‐G) Representative fluorescence pictures (F) and peak graphs of the linear ROI (G) demonstrating the colocalization of circTACC3 (red), S9.6‐indicated R loops (green), and NONO/p54 nrb (yellow) in nuclei (blue) with or without Dox‐inducible RNase H1 expression in PA and OA treated HCCLM3 and HepG2 cells. Abbreviations: MASH, metabolic dysfunction‐associated steatohepatitis; HCC, hepatocellular carcinoma; PA, palmitic acid; OA, oleic acid; T, tumor tissue; PT, peritumoral normal tissue; SAH, S‐adenosylhomocysteine; DAA, 3‐deazaadenosine; DRIP, DNA‐RNA immunoprecipitation; Dox, Doxycycline; ROI, region of interest.

Article Snippet: Immunocomplexes were incubated with the fluorescein‐conjugated secondary antibody (# C61012 ‐05, mouse, Li‐Cor, NE, US; # C80416 ‐08, rabbit, Li‐Cor) and then detected using the Odyssey fluorescence scanner (Li‐Cor).

Techniques: Modification, Staining, Derivative Assay, Fluorescence, Transfection, Fractionation, Expressing, RNA Immunoprecipitation

Journal: Cancer Communications

Article Title: Intranuclear paraspeckle‐circular RNA TACC3 assembly forms RNA‐DNA hybrids to facilitate MASH‐related hepatocellular carcinoma growth in an m 6 A‐dependent manner

doi: 10.1002/cac2.70061

Figure Lengend Snippet: DSB‐circTACC3‐R loops aggregated to promote the inter‐TADs contact. (A) After DRIP‐ChIRP sequencing, the reads distributions across peaks of all independent biological replicates are presented. (B) Genome‐wide distribution of the circTACC3‐R Loop‐located genes positively correlated with PA + OA induction or negatively correlated with m 6 A modification intervention. (C) Representative fluorescence images of the colocalization of indicated molecules in nuclei of HCC cells treated with or without PA and OA following RNase R treatment. (D) Schematic representation shows combination of DRIP‐ChIRP‐seq and γH2AX CUT&Tag‐seq to analyze the distribution of the DSB‐circTACC3‐R Loop structures in the genome. (E) Genome‐wide distribution of the DSB‐circTACC3‐R loop located genes in PA + OA induced HepG2 cells. (F) Top four enriched DSB‐circTACC3‐R loop‐binding motifs based on de novo motif analysis. (G) The dynamic clustering of paraspeckles (indicated by NONO/p54 nrb ‐mCherry) were filmed using STELLARIS Dynamic Signal Enhancement 24 h after PA + OA induction at 5‐min intervals for a duration of 1.5 h. Examples (from 50 min to 85 min) of fusions of several NONO/p54 nrb ‐mCherry foci are shown (time points indicated in minutes). (H) Heatmap depicting the fold change(log 2 ) in Hi‐C contact frequencies between PA + OA‐treated and control cells throughout chromosome 7. Interactions that increase in PA + OA group (red) or decrease in Mock group (blue) are evident. Profile of DRIP‐ChIRP‐seq and γH2AX CUT&Tag‐seq are shown on the top. TADs that had higher inter‐TADs contact frequencies (named “contact‐elevated TADs”) in both long‐range (green box) and between adjacent TADs (red box) are marked. DSB‐circTACC3‐R loops are marked with red arrow. (I) Hi‐C maps around the human STX6 locus that formed DSB‐circTACC3‐R loop structure are shown. DSB‐circTACC3‐R loops are marked with red arrow. Abbreviations: DRIP, DNA‐RNA immunoprecipitation; ChIRP, chromatin isolation by RNA purification; γH2AX, Ser‐139 residue of the histone variant H2AX; CUT&Tag, cleavage under targets and tagmentation; IF, immunofluorescence; FISH, fluorescence i n situ hybridization; PA, palmitic acid; OA, oleic acid; Hi‐C, high‐throughput/resolution chromosome conformation capture; DSB, DNA double‐strand breaks; STX6, Syntaxin 6.

Article Snippet: Immunocomplexes were incubated with the fluorescein‐conjugated secondary antibody (# C61012 ‐05, mouse, Li‐Cor, NE, US; # C80416 ‐08, rabbit, Li‐Cor) and then detected using the Odyssey fluorescence scanner (Li‐Cor).

Techniques: Sequencing, Genome Wide, Modification, Fluorescence, Binding Assay, Hi-C, Control, RNA Immunoprecipitation, Isolation, Purification, Residue, Variant Assay, Immunofluorescence, Hybridization, High Throughput Screening Assay

Journal: Cancer Communications

Article Title: Intranuclear paraspeckle‐circular RNA TACC3 assembly forms RNA‐DNA hybrids to facilitate MASH‐related hepatocellular carcinoma growth in an m 6 A‐dependent manner

doi: 10.1002/cac2.70061

Figure Lengend Snippet: DSB‐circTACC3‐R loop‐localized genes are selectively activated. (A) List of DSB‐circTACC3‐R loop‐localized genes. (B) DSB‐circTACC3‐loop‐localized genes expression in HepG2 cells with/without lipid overload induction ( n = 4). * P < 0.05; ** P < 0.01; *** P < 0.001; NS , not significant. (C‐D) DRIP‐ChIRP‐seq (C) and γH2AX CUT&Tag‐seq (D) RPKM analysis of DSB‐circTACC3‐loop‐localized genes to compare circTACC3‐R Loop enrichment within the “contact‐elevated TADs” ( n = 26) or not within the “contact‐elevated TADs” ( n = 12). * P < 0.05; ** P < 0.01; *** P < 0.001; NS , not significant. (E‐F) Representative fluorescence images (E) and peaks graphs of the linear ROI (F) show the colocalization of indicated molecules with or without Dox‐inducible RNase H1 expression. (G) NONO/p54 nrb ‐mCherry HepG2 cells with or without Dox‐inducible RNase H1 expression were filmed 24 h after PA + OA induction at 5‐min intervals. Abbreviations: PA, palmitic acid; OA, oleic acid; TAD, topologically associated domain; γH2AX, Ser‐139 residue of the histone variant H2AX; CUT&Tag, cleavage under targets and tagmentation; RPKM, reads per kilobase per million mapped reads; Dox, Doxycycline; ROI, region of interest.

Article Snippet: Immunocomplexes were incubated with the fluorescein‐conjugated secondary antibody (# C61012 ‐05, mouse, Li‐Cor, NE, US; # C80416 ‐08, rabbit, Li‐Cor) and then detected using the Odyssey fluorescence scanner (Li‐Cor).

Techniques: Expressing, Fluorescence, Residue, Variant Assay

Journal: Frontiers in Molecular Biosciences

Article Title: A systematic review of the barcoding strategy that contributes to COVID-19 diagnostics at a population level

doi: 10.3389/fmolb.2023.1141534

Figure Lengend Snippet: Schematic representation of mechanistic strategies of barcoding. (A–C) Barcodes can be introduced to a template using adaptors through direct ligation (A) , using RT- or PCR primers at the reverse transcription or PCR amplification step (B) , and using hybridizing molecular inversion probes (C) . (D) Schematic representation of the difference between “barcodes” and “sample indexes”. Barcodes aim to correct sequencing errors. For example, a misreading nucleotide, guanosine (G) can be corrected in final consensus sequences for a pool of Sample 1 (top panel). Sample indexes are used to multiplex different sequencing amplicons generated from different pools of samples (Sample 1, 2, and 3) (bottom panel). Panel (A) is modified based on in and panel (C) is modified based on in .

Article Snippet: Primer-associated approach , Sequence-based barcodes , SQK-RBK004: transposase carrying barcodes to the site of the cleavage , - , - , Whole genome , Oxford Nanopore Rapid Barcoding kit (SQK-RBK004) , SARS-CoV-2 patient samples (nasopharyngeal swab) , Oxford Nanopore , Guppy version 3.6.0; ARTIC Network bioinformatics protocol , Multiplex samples , Propose a method to sequence the whole genome of SARS-CoV-2 in a rapid and cost-efficient manner , .

Techniques: Ligation, Reverse Transcription, Amplification, Sequencing, Multiplex Assay, Generated, Modification

Journal: Frontiers in Molecular Biosciences

Article Title: A systematic review of the barcoding strategy that contributes to COVID-19 diagnostics at a population level

doi: 10.3389/fmolb.2023.1141534

Figure Lengend Snippet: Systematic comparison of barcoding strategies used in the category of molecular barcodes.

Article Snippet: Primer-associated approach , Sequence-based barcodes , SQK-RBK004: transposase carrying barcodes to the site of the cleavage , - , - , Whole genome , Oxford Nanopore Rapid Barcoding kit (SQK-RBK004) , SARS-CoV-2 patient samples (nasopharyngeal swab) , Oxford Nanopore , Guppy version 3.6.0; ARTIC Network bioinformatics protocol , Multiplex samples , Propose a method to sequence the whole genome of SARS-CoV-2 in a rapid and cost-efficient manner , .

Techniques: Comparison, Software, Sequencing, Multiplex Assay, CRISPR, Plasmid Preparation, Microarray, Binding Assay, Amplification, Extraction, Ligation, DNA Sequencing, Multiplexing, Generated, Reverse Transcription, Staining, Flow Cytometry, High Throughput Screening Assay, Inhibition, Blocking Assay, Conjugation Assay, RNA Sequencing Assay, Transmission Assay, Incubation, Diagnostic Assay, Next-Generation Sequencing, Infection

Journal: Cell host & microbe

Article Title: NON-NEUTRALIZING ANTIBODIES FROM A MARBURG INFECTION SURVIVOR MEDIATE PROTECTION BY FC-EFFECTOR FUNCTIONS AND ENHANCING EFFICACY OF OTHER ANTIBODIES

doi: 10.1016/j.chom.2020.03.025

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse anti-human IgG3 Hinge-AP , Southern Biotech , Cat#9210-04; RRID: AB_2687998.

Techniques: Recombinant, Virus, Clinical Proteomics, Infection, Staining, Expressing, Saline, Sequencing, Plasmid Preparation, Mutagenesis, Ligation, One Step RT-PCR, Peptide Microarray, Software, Flow Cytometry, High Throughput Screening Assay, Cytometry, Chromatography, Microscopy, Magnetic Beads

Journal: BMC Cancer

Article Title: RNF43 ubiquitinates and degrades phosphorylated E-cadherin by c-Src to facilitate epithelial-mesenchymal transition in lung adenocarcinoma

doi: 10.1186/s12885-019-5880-1

Figure Lengend Snippet: RNF43 was associated with E-cadherin downregulation in lung adenocarcinoma. a , Total protein extracts from cancerous (left) and paracancerous (right) tissues were analyzed by antibodies and processed for high-throughput immunoblotting using protein antibody microarrays consisting of 650 antibodies. b , The microarray analysis described in A unveiled the differences in protein expression between cancerous and paracancerous tissues, revealing significant upregulation (red color) of RNF43 and p-Src in lung adenocarcinoma. The green and yellow colors represent equal signals and downregulation (E-cad) in lung adenocarcinoma. c , Luciferase-based siRNA library screen for human E3 ligases identified multiple E3 candidates upon knockdown in SUM-E-cad-Luc/R-Luc cells, which increased luciferase activity by more than 4-fold. d , Immunohistochemical staining for RNF43 and E-cadherin was performed at high magnification (× 400)

Article Snippet: Total cell lysates (TCLs) were used for immunoprecipitation using an anti-E-cadherin antibody (ab-1416 Abcam), in which ubiquitinated E-cadherin was detected by immunoblotting with an anti-ubiquitin antibody (ab-7780 Abcam).

Techniques: High Throughput Screening Assay, Western Blot, Microarray, Expressing, Luciferase, Activity Assay, Immunohistochemical staining, Staining

Journal: BMC Cancer

Article Title: RNF43 ubiquitinates and degrades phosphorylated E-cadherin by c-Src to facilitate epithelial-mesenchymal transition in lung adenocarcinoma

doi: 10.1186/s12885-019-5880-1

Figure Lengend Snippet: Activated c-Src mediated the ubiquitination and degradation of E-cadherin in lung adenocarcinoma cells. a , Immunoblot analysis of c-Src, pY416-Src (p-Src), E-cadherin (E-cad), RNF43, Hakai and β-actin in the human lung adenocarcinoma cell lines A549, NCI-H522, NCI-H1975, NCI-H1623 and NCI-H2073. b , A549 cells were stably transfected with lentivirus containing control or Caspase-8 shRNA (Cap8 KD). Caspase-8, p-Src, c-Src, E-cad and β-actin in total cell lysates (TCLs) were detected by western blot. c , Caspase-8-deficient H522 cells were stably transfected with a control vector or wild-type Caspase-8 via adenovirus. Caspase-8, p-Src, c-Src, E-cad and β-actin in TCLs were detected by western blot. d , The TCLs of A549 and H522 cells in which c-Src was activated were subjected to immunoprecipitation (IP) using an anti-E-cadherin antibody. Ubiquitinated (Ubi-) E-cadherin was detected by western blot ( upper blot ). The blot was then stripped and reprobed for RNF43 ( second from upper panel ). RNF43, E-cadherin and β-actin were in TCLs detected by western blot ( lower blot ). e , A549 cells were allowed to attach to dishes for 12 h and then assessed by confocal microscopy using antibodies against E-cadherin and RNF43. Scale bars, 10 μm. f , E-cadherin mRNA was examined in A549 + control/Caspase-8 shRNA cells (Casp8 KD) and in H522 + control vector/wild-type Caspase-8 cells

Article Snippet: Total cell lysates (TCLs) were used for immunoprecipitation using an anti-E-cadherin antibody (ab-1416 Abcam), in which ubiquitinated E-cadherin was detected by immunoblotting with an anti-ubiquitin antibody (ab-7780 Abcam).

Techniques: Western Blot, Stable Transfection, Transfection, shRNA, Plasmid Preparation, Immunoprecipitation, Confocal Microscopy

Journal: BMC Cancer

Article Title: RNF43 ubiquitinates and degrades phosphorylated E-cadherin by c-Src to facilitate epithelial-mesenchymal transition in lung adenocarcinoma

doi: 10.1186/s12885-019-5880-1

Figure Lengend Snippet: RNF43-induced E-cadherin ubiquitination and degradation initiated EMT phenotype via allowing β-catenin to translocate into the nucleus in a c-Src-dependent manner. a , A549 cells were stably transfected with the lentiviral control shRNA, RNF43 shRNA, or c-Src shRNA. Snail, Slug, Twist, E-cadherin (E-cad), Vimentin, Fibronectin, N-cadherin (N-cad), and β-actin in total cell lysates (TCLs) were detected by western blot. b , A549 cells stably transfected with lentiviral control shRNA, RNF43 shRNA, or c-Src shRNA were seeded overnight. TCLs were subjected to immunoprecipitation (IP) using an anti-E-cadherin antibody. Ubiquitinated E-cadherin was detected by western blot with an anti-ubiquitin antibody ( upper blot ). Phospho-β-catenin (p-β-catenin), β-catenin, GSK-3β, RNF43, and β-actin in TCLs were detected by western blot ( middle blot ). β-catenin was detected in the nuclear extract of A549 cells ( lower blot ). c , The TCLs of A549 cells stably transfected with lentiviral control shRNA, RNF43 shRNA, or c-Src shRNA were subjected to IP using an anti-β-catenin antibody. E-cadherin, GSK-3β, TCF-4, and β-catenin were detected by western blot. d , A549 cells were treated with an shRNA either not targeting TCF-4 (Control) or targeting TCF-4 (TCF-4). The in vitro β-catenin antibody 7A7 was delivered into A549 cells. Immunoblot analysis of LaminA/C (loading control), TCF-4, E-cad, Vimentin, and nuclear/total β-catenin was performed using appropriate antibodies. e , A549 cells were treated with an shRNA either not targeting (Control) or targeting TCF-4 (TCF-4). The in vitro β-catenin antibody 7A7 was delivered into A549 cells. The mRNA levels of E-cad, Vimentin, and RNF43 were examined by RT-PCR. GAPDH mRNA was used to confirm equal loading. f and g , β-catenin and TCF-4 chromatin immunoprecipitation (ChIP) in A549 cells. PCR was carried out using primers specific for the promoter regions of E-cad, Vimentin, and RNF43. One-fifth of the input DNA from each sample was also amplified and designated as Input. h , Schematic representation of reporter plasmids containing the control vector, E-cadherin promoter, Vimentin promoter, Vimentin promoter with WRE deletion (Vim mut), RNF43 promoter, and RNF43 promoter with WRE deletion (RNF43 mut). i , A549 cells were transiently transfected with the reporter plasmids described in f . Luciferase activities were measured in triplicate (mean ± SD, t test, *, P < 0.05)

Article Snippet: Total cell lysates (TCLs) were used for immunoprecipitation using an anti-E-cadherin antibody (ab-1416 Abcam), in which ubiquitinated E-cadherin was detected by immunoblotting with an anti-ubiquitin antibody (ab-7780 Abcam).

Techniques: Stable Transfection, Transfection, shRNA, Western Blot, Immunoprecipitation, In Vitro, Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation, Amplification, Plasmid Preparation, Luciferase

Journal: BMC Cancer

Article Title: RNF43 ubiquitinates and degrades phosphorylated E-cadherin by c-Src to facilitate epithelial-mesenchymal transition in lung adenocarcinoma

doi: 10.1186/s12885-019-5880-1

Figure Lengend Snippet: c-Src underlay RNF43-mediated E-cadherin ubiquitination through E-cadherin phosphorylation at tyrosine 797. a , H522 cells were stably transfected with wild-type c-Src (Src WT) and a constitutively active c-Src, Src Y527F. p-Src, c-Src, E-cadherin (E-cad), Vimentin, RNF43, total β-catenin, and β-actin via adenovirus and detected by western blot analysis of total cell lysates (TCLs) ( Upper blot ). β-catenin in the nuclear extract was evaluated using an antibody against β-catenin ( lower blot ). b , TCLs were subjected to immunoprecipitation (IP) using an anti-E-cadherin antibody. Ubiquitinated E-cadherin was detected with an anti-ubiquitin antibody. p-Src, RNF43, nonspecific phosphorylated tyrosine of E-cadherin (p-Tyr-100), and E-cadherin in the immunocomplex were detected by western blot. c , EMT morphological changes and metastatic incidences in H522 cells transfected with Src WT and Src Y527F ( χ 2 test, *, P < 0.05). Scale bars, 50 μm. d , Tyrosine 797 of E-cadherin was conserved across various species ( upper panel ). MALDI-mass spectrometry analysis of CNBr-cleaved endogenous E-cadherin expression in A549 cells with or without endogenous c-Src ( lower panel ). e , E-cadherin-knockdown A549 cells transfected with unmutated E-cadherin (E-cad WT) or E-cadherin mutated at Y755-757F (E-cad Y755-757F) or Y797F (E-cad Y797F) were lysed to obtain TCLs. E-cadherin (anti-HA) was immunoprecipitated from TCLs and probed for ubiquitin-E-cadherin, p-Src, nonspecific phosphorylated tyrosine of E-cadherin, RNF43, and E-cadherin (anti-HA) by western blot. f , E-cadherin-knockdown A549 cells transfected with HA-tagged E-cad WT, E-cad Y755-757F, or E-cad Y797F were lysed to obtain TCLs. p-Src, c-Src, Vimentin, E-cadherin (anti-HA), RNF43, total β-catenin, and β-actin were detected by western blot analysis of the TCLs ( upper blot ). Nuclear extracts were subjected to western blot analysis of β-catenin ( lower blot ). g , EMT morphological changes and metastatic incidences in E-cadherin-knockdown cells transfected with HA-tagged E-cad WT, E-cad Y755-757F, or E-cad Y797F ( χ 2 test, *, P < 0.05). Scale bars, 50 μm

Article Snippet: Total cell lysates (TCLs) were used for immunoprecipitation using an anti-E-cadherin antibody (ab-1416 Abcam), in which ubiquitinated E-cadherin was detected by immunoblotting with an anti-ubiquitin antibody (ab-7780 Abcam).

Techniques: Stable Transfection, Transfection, Western Blot, Immunoprecipitation, Mass Spectrometry, Expressing

Journal: BMC Cancer

Article Title: RNF43 ubiquitinates and degrades phosphorylated E-cadherin by c-Src to facilitate epithelial-mesenchymal transition in lung adenocarcinoma

doi: 10.1186/s12885-019-5880-1

Figure Lengend Snippet: Frz8 was required for the RNF43-induced ubiquitination of phosphorylated E-cadherin. a , Immunoblot analysis of Frz8, Frz2, Dishevelled 3 (Dvl3), LGR6, and β-actin in A549 and H522 cells. b , A549 and H522 cells stably transfected with control shRNA or RNF43 shRNA (RNF43 KD) via adenovirus were seeded overnight. The total cell lysates (TCLs) were subjected to immunoprecipitation (IP) using an anti-Frz8 antibody. Ubiquitinated Frz8 was detected by western blot with an anti-ubiquitin antibody ( upper blot ). Frz8 and RNF43 in the TCLs were detected by western blot ( lower blot ). c , A549 cells stably transfected with control shRNA or Frz8 shRNA (Frz8 KD) via lentivirus were seeded overnight. The TCLs were subjected to IP using an anti-E-cadherin antibody. Ubiquitinated E-cadherin was detected by western blot with an anti-ubiquitin antibody ( upper blot ). E-cadherin, Frz8, and β-actin in the TCLs were detected by western blot ( lower blot ). d , Schematic representation of reporter plasmids containing full-length RNF43 and two truncated versions of RNF43 as shown. e , RNF43-knockdown A549 cells stably transfected with RNF43, RNF43-R, or RNF43-P via adenovirus were seeded overnight. The TCLs were subjected to IP using anti-E-cadherin and anti-HA antibodies. Ubiquitinated E-cadherin, E-cadherin, Frz8, and HA were detected by western blot ( upper and middle blot ). E-cadherin and β-actin in the TCLs were detected by western blot ( lower blot ). f , A549 cells treated with DMSO (Control) or monoclonal antibodies (10.0 mmol/L) against the cysteine-rich domain (CRD) of Frz8 and the RNF43 protease domain (PA) were seeded overnight. The TCLs were subjected to IP using an anti-E-cadherin antibody. Ubiquitinated E-cadherin, RNF43, and E-cadherin were detected by western blot ( upper blot ). E-cadherin, Vimentin, and β-actin in the TCLs were detected by western blot ( middle blot ). β-catenin was detected in the nuclear extract ( lower blot ). g , A549 cells treated with DMSO (Control) or monoclonal antibodies (10.0 mmol/L) against the cysteine-rich domain (CRD) of Frz8 and the RNF43 protease domain (PA) were seeded overnight. The TCLs were subjected to IP using an anti-RNF43 antibody. Frz8, RNF43, and E-cadherin were detected by western blot. h , EMT morphological changes and metastatic incidences in A549 cells treated with DMSO (Control) or monoclonal antibodies (10.0 mmol/L) against the cysteine-rich domain (CRD) of Frz8 and the RNF43 protease domain (PA) ( χ 2 test, *, P < 0.05). Scale bars, 50 μm. i , A549 cells treated with DMSO (Control) and RSOP1–4 were seeded overnight. The TCLs were subjected to IP using anti-E-cadherin and anti-Frz8 antibodies. Ubiquitinated E-cadherin and RNF43 were detected by western blot ( upper and middle blot ). E-cadherin and β-actin in the TCLs were detected by western blot ( lower blot )

Article Snippet: Total cell lysates (TCLs) were used for immunoprecipitation using an anti-E-cadherin antibody (ab-1416 Abcam), in which ubiquitinated E-cadherin was detected by immunoblotting with an anti-ubiquitin antibody (ab-7780 Abcam).

Techniques: Western Blot, Stable Transfection, Transfection, shRNA, Immunoprecipitation

Journal: Human Molecular Genetics

Article Title: Altered keratinocyte differentiation is an early driver of keratin mutation-based palmoplantar keratoderma

doi: 10.1093/hmg/ddz050

Figure Lengend Snippet: Analysis of global gene expression in Krt16-null footpad lesions and comparison to human PC cases. (A) Volcano plot depicting differentially expressed genes in Krt16-null paw skin lesions relative to WT controls. (B) Top 10 genes differentially regulated in Krt16-null paw skin from established paw lesions compared to WT littermate controls. (C) Validation of downregulated genes identified by microarray analysis by RT-qPCR in Krt16-null paw skin lesions. N = 4 mice/genotype. Error bars are SEM. *P < 0.05, **P < 0.01. (D) Overlap in the significantly changed genes that are upregulated between Krt16-null paw skin lesions and KRT16 human cases. (E) Overlap in the significantly changed genes that are downregulated between Krt16-null paw skin lesions and KRT16 human cases. (F) Overlap in the significantly changed genes that are upregulated between Krt16-null paw skin lesions and KRT6 human cases. (G) Overlap in the significantly changed genes that are downregulated between Krt16-null paw skin lesions and KRT6 human cases. Boxes for (D)–(G) list common genes between mouse and human data sets (listed in alphabetical order).

Article Snippet: Total RNA isolated from paw skin, epidermis and dermis, of 2 month old Krt16 -null (2 males, 2 females) and WT (2 males, 2 females) littermates (see Biochemical and morphological analyses ) was subjected to microarray analysis by the Johns Hopkins Deep Sequencing Microarray Core using the Affymetrix Mouse Transcriptome Array 1.0 GeneChip.

Techniques: Gene Expression, Comparison, Biomarker Discovery, Microarray, Quantitative RT-PCR

Journal: Oncotarget

Article Title: By inhibiting snail signaling and miR-23a-3p, osthole suppresses the EMT-mediated metastatic ability in prostate cancer

doi:

Figure Lengend Snippet: A. A schematic representation of the procedure for miRNA selection. Differential expressions of miRNAs in osthole-treated cells versus vehicle-treated cells were analyzed with a OneArray ® miRNA profiling chip. B. Treatment of Du145 cells with osthole for 6 h. miR-146a, miR-22-3p, and miR-23a-3p expressions were detected by a quantitative PCR. C. Upper panel, Du145 cells were transfected with an miR-23a-3p mimic or mimic control for 24 h followed by osthole (60 μM) treatment for an additional 24 h. E-cadherin expression levels were determined by a Western blot analysis. Quantitative E-cadherin protein levels were adjusted to the β-actin protein level. Lower panel, Relative luciferase activities of DU145 cells co-transfected with an E-cadherin luciferase 3′UTR reporter vector and miR-23a-3p mimic or mimic control for 24 h. Values are presented as the mean ± SE of three independent experiments. * p < 0.05, compared to the control groups. D. DU145 cells were transfected with an miR-23a-3p mimic or mimic control for 24 h followed by osthole (60 μM) treatment for an additional 24 h. The cell-invasion ability was determined by a Matrigel invasion assay. Values are presented as the mean ± SE of three independent experiments. Data were analyzed using a one-way ANOVA with Tukey's post-hoc tests at 95% confidence intervals; different letters represent different levels of significance. E. DU145 cells were transfected with either an miR-23a-3p inhibitor or a negative control. The cell-invasion ability was determined by a Matrigel invasion assay. Values are presented as the mean ± SE of three independent experiments. * p < 0.05, compared to the control groups. F. PC3 or DU145 cells were treated with TGF-β for 6, 12, or 24 h. Expression levels of E-cadherin and miR-23a-3p were determined by Western blotting (upper panel) and a quantitative PCR (lower panel), respectively. Quantitative E-cadherin protein levels were adjusted to the β-actin protein level.

Article Snippet: To identify which miRNAs are regulated by osthole, a high-throughput and specific miRNA microarray (human miRNA OneArray ® miRNA profiling chip) using Du145 cells after osthole treatment was conducted by the Phalanx Biotech Group (Hsinchu, Taiwan).

Techniques: Selection, Real-time Polymerase Chain Reaction, Transfection, Expressing, Western Blot, Luciferase, Plasmid Preparation, Invasion Assay, Negative Control

Journal: Science immunology

Article Title: Core-binding factor β and Runx transcription factors promote adaptive natural killer cell responses

doi: 10.1126/sciimmunol.aan3796

Figure Lengend Snippet: (A) Graph shows mRNA expression of highest induced lymphocyte-specific transcription factors in splenic Ly49H+ NK cells sorted from uninfected and MCMV-infected animals on day 2 PI, as assessed by microarray [data provided by the Immunological Genome Consortium (41)]. Data are shown as fold change in microarray signal intensity for the infected versus uninfected samples (n = 3 biological replicates per group and representative of three separate experiments). Solid black bars denote significant up-regulation or down-regulation as compared with uninfected controls (P < 0.05, two-tailed unpaired Student’s t test). (B) Normalized counts of Runx1, Runx2, Runx3, and Cbfb in splenic Ly49H+ NK cells sorted from MCMV-infected mice on day 2 PI and uninfected mice (top) or in unstimulated (US) or IL-12 plus IL-18-treated (12 + 18; 16-hour stimulation) splenic NK cells (bottom), as assessed by RNA-seq (n = 2 to 3 biological replicates per group). (C) RNA-seq was performed on purified Ly49H+ WT NK cells and Stat4−/− NK cells from uninfected and MCMV-infected mixed BMC mice (day 2 PI). Normalized counts of Runx family members are shown (n = 2 to 3 biological replicates per group). (D) RNA-seq was performed on purified Ly49H+ and Ly49H− WT NK cells from uninfected and MCMV-infected mice (day 2 PI). Normalized counts of Runx1 and Runx3 are shown (n = 2 to 3 biological replicates per group). Data are presented as means ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: ChIP was performed as previously described ( 10 , 16 ), using 10 μg of rabbit polyclonal anti-STAT4 antibody (Santa Cruz Biotechnology, sc-468, clone C-20) or 1 μg of rabbit polyclonal anti-trimethyl-histone H3 (Lys4) antibody (H3K4me3; Millipore, 07473) followed by Illumina next-generation sequencing.

Techniques: Expressing, Infection, Microarray, Two Tailed Test, RNA Sequencing, Purification

Journal: Science immunology

Article Title: Core-binding factor β and Runx transcription factors promote adaptive natural killer cell responses

doi: 10.1126/sciimmunol.aan3796

Figure Lengend Snippet: Splenic NK cells (TCRβ−CD19−CD3ε−Ly6G−TER119−TCRγδ−NK1.1+) were sorted from WT mice and stimulated with IL-12 and IL-18 or media alone as a control (unstimulated). STAT4 ChIP was performed, followed by high-throughput DNA sequencing. (A) Proportions of STAT4 genome-wide occupancy at promoter (2 kb upstream and 0.5 kb downstream from TSS), intronic, exonic, or distal intergenic regions in cytokine-stimulated NK cells are shown. (B) RNA-seq was performed on splenic Ly49H+ WT NK cells and Stat4−/− NK cells sorted from mixed chimeras 2 days after MCMV infection. Venn diagram of overlap between differentially expressed genes (top; Padj < 0.05) identified through RNA-seq and reproducible STAT4-bound regions identified through ChIP-seq (bottom; IDR < 0.05). RNA-seq data were performed on n = 3 per condition. (C) Bar graphs depict the top 20 genes with greatest fold enrichment of STAT4 binding over input calculated by MACS2 in transcription factors that show differential expression in RNA-seq data. (D) Representative gene tracks for indicated core-binding factors from STAT4 ChIP-seq. ChIP-seq data are representative of three independent experiments with n = 15 to 20 pooled mice per group per experiment.

Article Snippet: ChIP was performed as previously described ( 10 , 16 ), using 10 μg of rabbit polyclonal anti-STAT4 antibody (Santa Cruz Biotechnology, sc-468, clone C-20) or 1 μg of rabbit polyclonal anti-trimethyl-histone H3 (Lys4) antibody (H3K4me3; Millipore, 07473) followed by Illumina next-generation sequencing.

Techniques: Control, High Throughput Screening Assay, DNA Sequencing, Genome Wide, RNA Sequencing, Infection, ChIP-sequencing, Binding Assay, Quantitative Proteomics

Journal: Science immunology

Article Title: Core-binding factor β and Runx transcription factors promote adaptive natural killer cell responses

doi: 10.1126/sciimmunol.aan3796

Figure Lengend Snippet: Splenic NK cells (TCRβ−CD19−CD3ε−Ly6G−TER119−TCRγδ−NK1.1+) were isolated from WT and Stat4−/− mice and stimulated with IL-12 and IL-18 or media alone as a control (unstimulated; unstim). H3K4me3 ChIP was performed, followed by high-throughput DNA sequencing. (A) Global proportions of H3K4me3 permissive marks at promoter, intronic, exonic, or distal inter-genic regions in cytokine-stimulated NK cells are shown. (B) Bar plots depict number of peaks that change on the basis of fold change (FC) of stimulated versus unstimulated NK cells. FC was calculated by taking the difference between log2-transformed normalized counts for each condition. Only peaks that showed a log2 FC greater than a magnitude of 1 were counted. (C) Meta-peak of all H3K4me3 promoter regions. Overlap of midpoints of ChIP fragments (defined as regions between properly paired sequence reads) for each TSS region was counted for each base pair ± 1 kb from the transcriptional start site. Line plot depicts average signal for all regions for each base pair. (D) Heat map of all H3K4me3 binding regions, with each row representing a single-peak region, row-clustered by normalized peak counts. Signal is displayed as normalized read counts over 5 kb centered at the peak region and is binned at 100-bp windows. (E) H3K4me3 signals from Cbfb, Runx1, Runx2, and Runx3 loci plotted as normalized fragment counts binned at 200 bp across a 10-kb window centered on the transcriptional start site. (F) Zoomed-in histograms of STAT4 ChIP and H3K4me3 ChIP reads mapped to Runx1 and Runx3 loci. Dashed box within boxed tracks indicate STAT4 ChIP called peak region. Data are representative of two independent experiments with n = 15 to 20 pooled mice per group per experiment.

Article Snippet: ChIP was performed as previously described ( 10 , 16 ), using 10 μg of rabbit polyclonal anti-STAT4 antibody (Santa Cruz Biotechnology, sc-468, clone C-20) or 1 μg of rabbit polyclonal anti-trimethyl-histone H3 (Lys4) antibody (H3K4me3; Millipore, 07473) followed by Illumina next-generation sequencing.

Techniques: Isolation, Control, High Throughput Screening Assay, DNA Sequencing, Transformation Assay, Sequencing, Binding Assay