monarch total rna miniprep kits  (New England Biolabs)


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    New England Biolabs monarch total rna miniprep kits
    Monarch Total Rna Miniprep Kits, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monarch total rna miniprep kits/product/New England Biolabs
    Average 98 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    monarch total rna miniprep kits - by Bioz Stars, 2022-08
    98/100 stars

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    New England Biolabs monarch total rna miniprep kit
    APB northern blot analysis of <t>tRNA</t> His GUG in control (WT) and siEhDUF2419 trophozoites that were co-cultivated with E. coli K12 or E. coli Δ QueC Control (WT) and siEhDUF2419 trophozoites were cultivated in the presence of E. coli K12 or E. coli Δ QueC for 7 days (ration of 1 trophozoite:1000 bacteria). (1) Wild-Type trophozoites (2) queuine-treated WT trophozoites (3) Wild-type trophozoites that were cultivated with E. coli K12 (4) Wild-type trophozoites that were cultivated with E. coli Δ QueC (5) siEhDUF2419 trophozoites (6) queuine-treated siEhDUF2419 trophozoites (7) siEhDUF2419 trophozoites that were cultivated with E. coli K12 (8) siEhDUF2419 trophozoites that were cultivated with E. coli Δ QueC (9) E. coli K12 <t>RNA.</t> The data represent two independent experiment that were repeated twice. p value
    Monarch Total Rna Miniprep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monarch total rna miniprep kit/product/New England Biolabs
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    monarch total rna miniprep kit - by Bioz Stars, 2022-08
    98/100 stars
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    APB northern blot analysis of tRNA His GUG in control (WT) and siEhDUF2419 trophozoites that were co-cultivated with E. coli K12 or E. coli Δ QueC Control (WT) and siEhDUF2419 trophozoites were cultivated in the presence of E. coli K12 or E. coli Δ QueC for 7 days (ration of 1 trophozoite:1000 bacteria). (1) Wild-Type trophozoites (2) queuine-treated WT trophozoites (3) Wild-type trophozoites that were cultivated with E. coli K12 (4) Wild-type trophozoites that were cultivated with E. coli Δ QueC (5) siEhDUF2419 trophozoites (6) queuine-treated siEhDUF2419 trophozoites (7) siEhDUF2419 trophozoites that were cultivated with E. coli K12 (8) siEhDUF2419 trophozoites that were cultivated with E. coli Δ QueC (9) E. coli K12 RNA. The data represent two independent experiment that were repeated twice. p value

    Journal: bioRxiv

    Article Title: Queuine salvaging in the human parasite Entamoeba histolytica

    doi: 10.1101/2022.06.21.496972

    Figure Lengend Snippet: APB northern blot analysis of tRNA His GUG in control (WT) and siEhDUF2419 trophozoites that were co-cultivated with E. coli K12 or E. coli Δ QueC Control (WT) and siEhDUF2419 trophozoites were cultivated in the presence of E. coli K12 or E. coli Δ QueC for 7 days (ration of 1 trophozoite:1000 bacteria). (1) Wild-Type trophozoites (2) queuine-treated WT trophozoites (3) Wild-type trophozoites that were cultivated with E. coli K12 (4) Wild-type trophozoites that were cultivated with E. coli Δ QueC (5) siEhDUF2419 trophozoites (6) queuine-treated siEhDUF2419 trophozoites (7) siEhDUF2419 trophozoites that were cultivated with E. coli K12 (8) siEhDUF2419 trophozoites that were cultivated with E. coli Δ QueC (9) E. coli K12 RNA. The data represent two independent experiment that were repeated twice. p value

    Article Snippet: RNA extraction using Monarch Total RNA Miniprep kit-total RNA was extracted from E. histolytica trophozoites that were incubated with E. coli K12/Δ QueC (a kind gift of Prof. Valérie de Crécy-Lagard, University of Florida, USA) using the Monarch Total RNA Miniprep Kit (NEW ENGLAND BioLabs) according to the manufacturer’s instruction.

    Techniques: Northern Blot

    Effect of BDF5 depletion on RNA levels and gene expression. a Flow cytometry of cells stained with SYTO RNASelect Stain to measure total RNA levels in Lmx::DiCre strains or the BDF5 −/+flx strain treated with rapamycin or DMSO over a 72 h time course. 20,000 events measured per condition. b Dot plot of total RNA-seq reads per protein-coding gene scaled to ERCC spike-in controls, then as a percentage of the DMSO control sample, separated per chromosome, conducted at a 96 h timepoint. Black lines denote the median of the scaled response for each chromosome, individual data points are means of 2 separate RNA seq experiments, the number of CDS features quantified on each chromosome is indicated above the dot plots. c Metaplot of divergent SSR ( n = 60) for DMSO treated or rapamycin-treated BDF5 −/+flx showing combined reads from the positive and negative strands. d . Metaplot of reads mapping to the + strand, normalised to ERCC control at divergent SSRs ( n = 60) of DMSO treated or rapamycin-treated BDF5 −/+flx cultures. e Metaplot of + stranded RNA-seq reads normalised to ERCC spike-in controls for PTUs ( n = 120), on a scale of 0–100%. f . Metaplot of reads mapping to the + and − strands, normalised to ERCC control at convergent SSRs ( n = 40) of DMSO treated or rapamycin-treated BDF5 −/+flx cultures. Metaplot data is from 1 representative of the three replicate RNA-seq datasets. g Spike-in controlled SYBR RT-qPCR of reporter genes for Pol I, II, III. BDF5 deletion was induced for 96 h and total RNA was extracted with lysis buffer spiked with yeast total RNA to provide a normalisation channel using a primer set against yeast actin, allowing comparison of the relative 18s rRNA, Cyclophilin A, and tRNA Lys RNA levels compared to DMSO treated cells. Bars denote mean, error bars denote standard deviation. Comparisons by multiple two-sided t test, corrected with Benjamini and Hochberg method, p-values indicate above, * denotes a discovery, n = 5 replicate PCR reactions. ACT1 values were not compared as this was the normalisation target.

    Journal: Nature Communications

    Article Title: Bromodomain factor 5 is an essential regulator of transcription in Leishmania

    doi: 10.1038/s41467-022-31742-1

    Figure Lengend Snippet: Effect of BDF5 depletion on RNA levels and gene expression. a Flow cytometry of cells stained with SYTO RNASelect Stain to measure total RNA levels in Lmx::DiCre strains or the BDF5 −/+flx strain treated with rapamycin or DMSO over a 72 h time course. 20,000 events measured per condition. b Dot plot of total RNA-seq reads per protein-coding gene scaled to ERCC spike-in controls, then as a percentage of the DMSO control sample, separated per chromosome, conducted at a 96 h timepoint. Black lines denote the median of the scaled response for each chromosome, individual data points are means of 2 separate RNA seq experiments, the number of CDS features quantified on each chromosome is indicated above the dot plots. c Metaplot of divergent SSR ( n = 60) for DMSO treated or rapamycin-treated BDF5 −/+flx showing combined reads from the positive and negative strands. d . Metaplot of reads mapping to the + strand, normalised to ERCC control at divergent SSRs ( n = 60) of DMSO treated or rapamycin-treated BDF5 −/+flx cultures. e Metaplot of + stranded RNA-seq reads normalised to ERCC spike-in controls for PTUs ( n = 120), on a scale of 0–100%. f . Metaplot of reads mapping to the + and − strands, normalised to ERCC control at convergent SSRs ( n = 40) of DMSO treated or rapamycin-treated BDF5 −/+flx cultures. Metaplot data is from 1 representative of the three replicate RNA-seq datasets. g Spike-in controlled SYBR RT-qPCR of reporter genes for Pol I, II, III. BDF5 deletion was induced for 96 h and total RNA was extracted with lysis buffer spiked with yeast total RNA to provide a normalisation channel using a primer set against yeast actin, allowing comparison of the relative 18s rRNA, Cyclophilin A, and tRNA Lys RNA levels compared to DMSO treated cells. Bars denote mean, error bars denote standard deviation. Comparisons by multiple two-sided t test, corrected with Benjamini and Hochberg method, p-values indicate above, * denotes a discovery, n = 5 replicate PCR reactions. ACT1 values were not compared as this was the normalisation target.

    Article Snippet: Total RNA was purified using NEB Monarch Total RNA MiniPrep Kit. cDNA was synthesised using NEB ProtoScript II with random hexamers.

    Techniques: Expressing, Flow Cytometry, Staining, RNA Sequencing Assay, Quantitative RT-PCR, Lysis, Standard Deviation, Polymerase Chain Reaction

    a) Distribution of annotated single hits over MEG3 gene, with statistically filtered EZH2-FLASH reads from two biological replicates in HUVECs. b) The occupancy of EZH2 hits over MEG3 features. Total reads per feature are given with exons being mostly occupies vs introns. c) Proportion of overlapping features over MEG3. The occupancy of EZH2 over each MEG3 exon is shown for two constitutively expressed transcripts. For both given transcripts there is high occupancy of exon 3. d) RNA immunoprecipitation (RIP) for EZH2 and H3K27me3 (repressive chromatin) followed by qPCR analysis. RIP-purified RNA from UV crosslinked HUVECs was used to prepare cDNA for qPCR analysis with primers against MEG3 (exon 3 region). Primers against U1snRNA gene serves as a negative control. Side diagram of EHZ2-MEG3 interacting region is charted as per FLASH hits and sequence. e) Distribution of EZH2 hybrids hits over MEG3 gene. Intermolecular MEG3-RNA interactions found in chimeras are captured by EZH2-FLASH-seq. Hits represent MEG3:MEG3 hybrids (black). IgG hybrids are plotted but are

    Journal: bioRxiv

    Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

    doi: 10.1101/2022.05.20.492787

    Figure Lengend Snippet: a) Distribution of annotated single hits over MEG3 gene, with statistically filtered EZH2-FLASH reads from two biological replicates in HUVECs. b) The occupancy of EZH2 hits over MEG3 features. Total reads per feature are given with exons being mostly occupies vs introns. c) Proportion of overlapping features over MEG3. The occupancy of EZH2 over each MEG3 exon is shown for two constitutively expressed transcripts. For both given transcripts there is high occupancy of exon 3. d) RNA immunoprecipitation (RIP) for EZH2 and H3K27me3 (repressive chromatin) followed by qPCR analysis. RIP-purified RNA from UV crosslinked HUVECs was used to prepare cDNA for qPCR analysis with primers against MEG3 (exon 3 region). Primers against U1snRNA gene serves as a negative control. Side diagram of EHZ2-MEG3 interacting region is charted as per FLASH hits and sequence. e) Distribution of EZH2 hybrids hits over MEG3 gene. Intermolecular MEG3-RNA interactions found in chimeras are captured by EZH2-FLASH-seq. Hits represent MEG3:MEG3 hybrids (black). IgG hybrids are plotted but are

    Article Snippet: The RNA Lysis was performed with above fractions using RNA Lysis Buffer (NEB, #T2012) and following the Monarch kit (NEB, #T2010).

    Techniques: Immunoprecipitation, Real-time Polymerase Chain Reaction, Purification, Negative Control, Sequencing

    a. Venn diagram showing the intersection between statistically filtered FLASH data from two biological replicates of our MEG3-ChIRP-seq-data (green), de novo hg38 analysed GEO RNA-seq data from siEZH2 deficient HUVECs (GSE71164, blue), and EZH2 ChIP-seq following MEG3 KD (yellow) and FLASH-seq transcriptome following EZH2 IP (pink). b. Correlation between gene expression levels and FLASH signal. Gray, expressed RefSeq genes with reproducible FLASH signal consistently detected in RNA-seq. Blue, genes with the highest RNA-seq signals and no reproducible FLASH signal belonging to integrin cell surface interaction pathway. Red , expressed ITGA4 gene, and green, ITGB1 gene, without reproducible FLASH signals. Data are from two biological replicates of each EZH2 FLASH sample and three biological replicates of EZH2 RNA-seq samples (Scr vs. siEZH2, GSE71164). c. Genomic tracks showing ChIRP-seq signal (MEG3 Odd, Even and LacZ) in HUVECs over ITGA4 gene only. The MEG3 binding site is located upstream of the ITGA4 gene in the promoter region, and it overlaps with the H3K27me3 signal and EZH2; as well as downstream within the ITGA4 gene body, where it overlaps with within the EZH2 signal in the intronic region of the gene. d. MEG3-ChIRP followed by qPCR, analysis of MEG3 binding region on ITGA4 in HUVECs. The crosslinked cell lysates were incubated with combined biotinylated probes against MEG3 lncRNA and the binding complexes recovered by magnetic streptavidin-conjugated beads. The qPCR was performed to detect the enrichment of specific region that associated with MEG3, peaks were related to input control and compared vs. the non-biotynilated control. e. ChIP-QPCR enrichment for EZH2 and H3K27me3 over ITGA4 promoter region in HUVECs depleted of MEG3 vs. Control.

    Journal: bioRxiv

    Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

    doi: 10.1101/2022.05.20.492787

    Figure Lengend Snippet: a. Venn diagram showing the intersection between statistically filtered FLASH data from two biological replicates of our MEG3-ChIRP-seq-data (green), de novo hg38 analysed GEO RNA-seq data from siEZH2 deficient HUVECs (GSE71164, blue), and EZH2 ChIP-seq following MEG3 KD (yellow) and FLASH-seq transcriptome following EZH2 IP (pink). b. Correlation between gene expression levels and FLASH signal. Gray, expressed RefSeq genes with reproducible FLASH signal consistently detected in RNA-seq. Blue, genes with the highest RNA-seq signals and no reproducible FLASH signal belonging to integrin cell surface interaction pathway. Red , expressed ITGA4 gene, and green, ITGB1 gene, without reproducible FLASH signals. Data are from two biological replicates of each EZH2 FLASH sample and three biological replicates of EZH2 RNA-seq samples (Scr vs. siEZH2, GSE71164). c. Genomic tracks showing ChIRP-seq signal (MEG3 Odd, Even and LacZ) in HUVECs over ITGA4 gene only. The MEG3 binding site is located upstream of the ITGA4 gene in the promoter region, and it overlaps with the H3K27me3 signal and EZH2; as well as downstream within the ITGA4 gene body, where it overlaps with within the EZH2 signal in the intronic region of the gene. d. MEG3-ChIRP followed by qPCR, analysis of MEG3 binding region on ITGA4 in HUVECs. The crosslinked cell lysates were incubated with combined biotinylated probes against MEG3 lncRNA and the binding complexes recovered by magnetic streptavidin-conjugated beads. The qPCR was performed to detect the enrichment of specific region that associated with MEG3, peaks were related to input control and compared vs. the non-biotynilated control. e. ChIP-QPCR enrichment for EZH2 and H3K27me3 over ITGA4 promoter region in HUVECs depleted of MEG3 vs. Control.

    Article Snippet: The RNA Lysis was performed with above fractions using RNA Lysis Buffer (NEB, #T2012) and following the Monarch kit (NEB, #T2010).

    Techniques: RNA Sequencing Assay, Chromatin Immunoprecipitation, Expressing, Binding Assay, Real-time Polymerase Chain Reaction, Incubation

    a ) RNA-seq dataset from HUVEC cells depleted in EZH2 (GSE71164) was de novo analysed and mapped onto Hg38 with reads given in the table. The principal component analysis (PCA) was used to describe the variance between two groups (ctr vs . siEZH2); depletion of EZH2 gene is represented between samples (n=3) with reads per sample, in the bottom table. b ) Heatmap of selected genes directly regulated by EZH2 and involved in angiogenesis and cell adhesion processes.

    Journal: bioRxiv

    Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

    doi: 10.1101/2022.05.20.492787

    Figure Lengend Snippet: a ) RNA-seq dataset from HUVEC cells depleted in EZH2 (GSE71164) was de novo analysed and mapped onto Hg38 with reads given in the table. The principal component analysis (PCA) was used to describe the variance between two groups (ctr vs . siEZH2); depletion of EZH2 gene is represented between samples (n=3) with reads per sample, in the bottom table. b ) Heatmap of selected genes directly regulated by EZH2 and involved in angiogenesis and cell adhesion processes.

    Article Snippet: The RNA Lysis was performed with above fractions using RNA Lysis Buffer (NEB, #T2012) and following the Monarch kit (NEB, #T2010).

    Techniques: RNA Sequencing Assay

    a) Computational analysis pipeline used to obtain orthologous peaks in human and intersect regions and genes enriched in repressive chromatin (H3K27me3) from ChIP-seq public dataset GSE114283. Up- and down-regulated genes were obtained associated with the peak region within 2000bp, and relevant function and biological pathway were associated using GREAT and DAVID analysis b) Overlap of the GEO datasets from a (Microarray GSE73524 ) and b (RNA-seq GSE71164 ) and the GSE114283 ChIP-seq reads of H3K27me 3 distribution in mouse MN cells depleted of MEG3 vs. control. ChIP extracted peaks unique to Ctrl vs. MEG3 KD were obtained, and associated mouse gene list composed based on reduction in H3K27me 3 signal. Using gene orthologous analysis in gProfiler we obtained human orthologous targets that was used for data intersection. c) Maximum peak scores of the overlapping signal over ITGA4 promoter, obtained by intersection of EZH2 ChIP signal with MEG3-ChIRP signal at this region. Upon depletion of MEG3 the EZH2 signal is significantly reduced whereby no overlap with MEG3 ChIRP signal is seen. d) Relative expression of ITGA4 in HUVEC measuring the levels of ITGA4 following addition of siRNA (50nM).

    Journal: bioRxiv

    Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

    doi: 10.1101/2022.05.20.492787

    Figure Lengend Snippet: a) Computational analysis pipeline used to obtain orthologous peaks in human and intersect regions and genes enriched in repressive chromatin (H3K27me3) from ChIP-seq public dataset GSE114283. Up- and down-regulated genes were obtained associated with the peak region within 2000bp, and relevant function and biological pathway were associated using GREAT and DAVID analysis b) Overlap of the GEO datasets from a (Microarray GSE73524 ) and b (RNA-seq GSE71164 ) and the GSE114283 ChIP-seq reads of H3K27me 3 distribution in mouse MN cells depleted of MEG3 vs. control. ChIP extracted peaks unique to Ctrl vs. MEG3 KD were obtained, and associated mouse gene list composed based on reduction in H3K27me 3 signal. Using gene orthologous analysis in gProfiler we obtained human orthologous targets that was used for data intersection. c) Maximum peak scores of the overlapping signal over ITGA4 promoter, obtained by intersection of EZH2 ChIP signal with MEG3-ChIRP signal at this region. Upon depletion of MEG3 the EZH2 signal is significantly reduced whereby no overlap with MEG3 ChIRP signal is seen. d) Relative expression of ITGA4 in HUVEC measuring the levels of ITGA4 following addition of siRNA (50nM).

    Article Snippet: The RNA Lysis was performed with above fractions using RNA Lysis Buffer (NEB, #T2012) and following the Monarch kit (NEB, #T2010).

    Techniques: Chromatin Immunoprecipitation, Microarray, RNA Sequencing Assay, Expressing

    a. Overview of the critical steps to obtain MEG3-bound genomic loci and intersections with EZH2 and H3K27me3 signals (obtained from GEO databases for HUVECs). In addition, enhancer regions were mapped within the genomic tracks. The intersection between GEO EZH2 ChIP data, GEO H3K27me3 ChIP data and statistically filtered MEG3-ChIRP data from two biological replicates was performed. The number of genes and degree of overlap is obtained between MEG3 and PRC2-dependent genes. The p-values are a result of hypergeometric test. b. Distribution of MEG3 peaks overlapping EZH2-ChIP peaks or H3K27me3-peaks with intersecting reads in relation to (i) gene regions and (ii) gene-type. c. Maximum peak score of ChIP signal for EZH2 and H3K27me3 intersecting the top enriched MEG3 peaks associated with nearest genes. Highest EZH2 peak score is over ITGA4, whereas H3K27me3 was detected in ITGA4, ITGA7, ITGA8 and ITGA9, members of ITGA family. d. Normalized reads from RNA-seq de novo analysis of GEO: GSE71164 dataset on Hg38, and expression of ITGA4 gene between Scr and siEZH2 depleted HUVECs, showing that ITGA4 is targeted by EZH2. Dataset in d and e is compared using Student’s t-test. e. ITGA4 expression from microarray analysis in C2C12 cells depleted of MEG3 (10nM, LNA GapMer) as per GEO dataset: GSE73524. The data shows that ITGA4 is a direct target of MEG3. f. (i) Total number of representable peaks (mRNA, antisense and lncRNA genes) from ChIP-seq analysis of Scr vs. MEG3 KD HUVECs. (ii ) Depletion of MEG3 gene in HUVECs (10nM LNA gapmers) was achieved with relative expression showing ∼70% reduction compared with Scr control. g. (i) Heat map showing distribution of reads and EZH2 densities at all unique RefSeq genes within TSSs ± 3 kb, sorted by EZH2 occupancy, in Control vs. MEG3 deficient (10nM) HUVECs. (ii) Overlap of ChIP-results between MEG3 and EZH2-dependent genes, with overlapped genes belonging to the biological pathway regulating cell adhesion. The common targets had lost or reduced EZH2 ChIP-signal.

    Journal: bioRxiv

    Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

    doi: 10.1101/2022.05.20.492787

    Figure Lengend Snippet: a. Overview of the critical steps to obtain MEG3-bound genomic loci and intersections with EZH2 and H3K27me3 signals (obtained from GEO databases for HUVECs). In addition, enhancer regions were mapped within the genomic tracks. The intersection between GEO EZH2 ChIP data, GEO H3K27me3 ChIP data and statistically filtered MEG3-ChIRP data from two biological replicates was performed. The number of genes and degree of overlap is obtained between MEG3 and PRC2-dependent genes. The p-values are a result of hypergeometric test. b. Distribution of MEG3 peaks overlapping EZH2-ChIP peaks or H3K27me3-peaks with intersecting reads in relation to (i) gene regions and (ii) gene-type. c. Maximum peak score of ChIP signal for EZH2 and H3K27me3 intersecting the top enriched MEG3 peaks associated with nearest genes. Highest EZH2 peak score is over ITGA4, whereas H3K27me3 was detected in ITGA4, ITGA7, ITGA8 and ITGA9, members of ITGA family. d. Normalized reads from RNA-seq de novo analysis of GEO: GSE71164 dataset on Hg38, and expression of ITGA4 gene between Scr and siEZH2 depleted HUVECs, showing that ITGA4 is targeted by EZH2. Dataset in d and e is compared using Student’s t-test. e. ITGA4 expression from microarray analysis in C2C12 cells depleted of MEG3 (10nM, LNA GapMer) as per GEO dataset: GSE73524. The data shows that ITGA4 is a direct target of MEG3. f. (i) Total number of representable peaks (mRNA, antisense and lncRNA genes) from ChIP-seq analysis of Scr vs. MEG3 KD HUVECs. (ii ) Depletion of MEG3 gene in HUVECs (10nM LNA gapmers) was achieved with relative expression showing ∼70% reduction compared with Scr control. g. (i) Heat map showing distribution of reads and EZH2 densities at all unique RefSeq genes within TSSs ± 3 kb, sorted by EZH2 occupancy, in Control vs. MEG3 deficient (10nM) HUVECs. (ii) Overlap of ChIP-results between MEG3 and EZH2-dependent genes, with overlapped genes belonging to the biological pathway regulating cell adhesion. The common targets had lost or reduced EZH2 ChIP-signal.

    Article Snippet: The RNA Lysis was performed with above fractions using RNA Lysis Buffer (NEB, #T2012) and following the Monarch kit (NEB, #T2010).

    Techniques: Chromatin Immunoprecipitation, RNA Sequencing Assay, Expressing, Microarray

    a. Schematic representation of steps in FLASH-seq (formaldehyde and UV cross-linking, ligation, a nd s equencing of h ybrids) with EZH2 immunoprecipitation using lysates from UV crosslinked endothelial cells. Dynamic EZH2-RNA complex formation occurs as represented. Following RNA ligation and chimera formation between interacting RNAs, sequencing is performed. Further analysis of single and hybrid reads bound by EZH2, reveals interacting RNA molecules. b. Distribution of annotated reads over genome, with gene classification (biotype), from statistically filtered EZH2-FLASH data with two biological replicates in HUVECs and MEG3-lncRNA (yellow wedge) as the candidate. c. I and ii Enriched motifs with sequences in MEG3 mRNA of EZH2-FLASH that uniquely overlap exons; the logos were drawn using the top 4-8nucleotides K-mers for each experimental replicate ( top and middle ) and z-score for each. Motif analysis was performed using the MEME suite (Bailey et al., 2009) [ 33 ] iii : Enriched motif within the fragments of MEG3:MEG3 hybrids d. Total RNA-RNA interactions associated with MEG3 at chr14:101292445-101327360, MEG3 id = NR_002766.2 ) and distribution of all MEG3 interactions among various classes of RNAs as captured by EZH2-FLASH. e. Intermolecular MEG3-RNA interactions found in chimeras captured by EZH2-FLASH. Chimera counts were mapped for all genomic features of annotated hybrids and the ones of MEG3 were plotted in the circos plot with position along the MEG3 genomic sequence. The main MEG3 hybrid is MEG3 and are represented by the number of interactions in red. The feature as a line: Red circle shows the position in the MEG3 gene in kilobases with * 50-55kb falling within exon3; Blue circle is a visual representation of MEG3 exons. Regions overlapping exons are represented in solid blue. Purple broad circle shows the nucleotides. The nucleotides at each position are: A : dark blue, C : light blue, T : light red, G : dark red. The details on the feature: The inner part of the white circle shows MEG3:MEG3 hybrids; Arcs connecting the centre of each hybrid fragment are shown in red, and the regions spanned by the hybrid fragments are shown in light green.

    Journal: bioRxiv

    Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

    doi: 10.1101/2022.05.20.492787

    Figure Lengend Snippet: a. Schematic representation of steps in FLASH-seq (formaldehyde and UV cross-linking, ligation, a nd s equencing of h ybrids) with EZH2 immunoprecipitation using lysates from UV crosslinked endothelial cells. Dynamic EZH2-RNA complex formation occurs as represented. Following RNA ligation and chimera formation between interacting RNAs, sequencing is performed. Further analysis of single and hybrid reads bound by EZH2, reveals interacting RNA molecules. b. Distribution of annotated reads over genome, with gene classification (biotype), from statistically filtered EZH2-FLASH data with two biological replicates in HUVECs and MEG3-lncRNA (yellow wedge) as the candidate. c. I and ii Enriched motifs with sequences in MEG3 mRNA of EZH2-FLASH that uniquely overlap exons; the logos were drawn using the top 4-8nucleotides K-mers for each experimental replicate ( top and middle ) and z-score for each. Motif analysis was performed using the MEME suite (Bailey et al., 2009) [ 33 ] iii : Enriched motif within the fragments of MEG3:MEG3 hybrids d. Total RNA-RNA interactions associated with MEG3 at chr14:101292445-101327360, MEG3 id = NR_002766.2 ) and distribution of all MEG3 interactions among various classes of RNAs as captured by EZH2-FLASH. e. Intermolecular MEG3-RNA interactions found in chimeras captured by EZH2-FLASH. Chimera counts were mapped for all genomic features of annotated hybrids and the ones of MEG3 were plotted in the circos plot with position along the MEG3 genomic sequence. The main MEG3 hybrid is MEG3 and are represented by the number of interactions in red. The feature as a line: Red circle shows the position in the MEG3 gene in kilobases with * 50-55kb falling within exon3; Blue circle is a visual representation of MEG3 exons. Regions overlapping exons are represented in solid blue. Purple broad circle shows the nucleotides. The nucleotides at each position are: A : dark blue, C : light blue, T : light red, G : dark red. The details on the feature: The inner part of the white circle shows MEG3:MEG3 hybrids; Arcs connecting the centre of each hybrid fragment are shown in red, and the regions spanned by the hybrid fragments are shown in light green.

    Article Snippet: The RNA Lysis was performed with above fractions using RNA Lysis Buffer (NEB, #T2012) and following the Monarch kit (NEB, #T2010).

    Techniques: Ligation, Immunoprecipitation, Sequencing

    a) Overview of the design of probes against MEG3 gene that were divided in probe Set1 and Set 2. The biotynilated probes were of 20 nucleotides and were spaced out 200 nucleotides apart down the gene length. b) Validation of MEG3 probes specifically binding MEG3 gene, by ChIRP-qPCR in HUVECs. Pull down with probe set 1 or set 2 retrieved 100% and 40% RNA, respectively. GAPDH primers were used as control and MEG3-associated samples did not amplify. c) Computational analysis pipeline for ChIRP-seq outlining data processing. The peak coverage was within the 100bp window. d) MEG3-ChIRP peaks associated with EZH2 gene as precipitated using both sets of probes (set 1 and 2). e) Enrichment of MEG3 signal by ChIRP-qpcr versus negative control (Background) at named promoter regions. MEG3 binding to genomic loci as validate by ChIRP-qPCR in HUVECs. Pull downs were performed with joined Odd and Even probes. Value 1 is a background level, defined by enrichment to LacZ negative probes in ChIRP. Control primers were designed for positive ChIRP peaks and used as a positive control and for regions deprived of MEG3-ChIRP reads as a negative control .

    Journal: bioRxiv

    Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

    doi: 10.1101/2022.05.20.492787

    Figure Lengend Snippet: a) Overview of the design of probes against MEG3 gene that were divided in probe Set1 and Set 2. The biotynilated probes were of 20 nucleotides and were spaced out 200 nucleotides apart down the gene length. b) Validation of MEG3 probes specifically binding MEG3 gene, by ChIRP-qPCR in HUVECs. Pull down with probe set 1 or set 2 retrieved 100% and 40% RNA, respectively. GAPDH primers were used as control and MEG3-associated samples did not amplify. c) Computational analysis pipeline for ChIRP-seq outlining data processing. The peak coverage was within the 100bp window. d) MEG3-ChIRP peaks associated with EZH2 gene as precipitated using both sets of probes (set 1 and 2). e) Enrichment of MEG3 signal by ChIRP-qpcr versus negative control (Background) at named promoter regions. MEG3 binding to genomic loci as validate by ChIRP-qPCR in HUVECs. Pull downs were performed with joined Odd and Even probes. Value 1 is a background level, defined by enrichment to LacZ negative probes in ChIRP. Control primers were designed for positive ChIRP peaks and used as a positive control and for regions deprived of MEG3-ChIRP reads as a negative control .

    Article Snippet: The RNA Lysis was performed with above fractions using RNA Lysis Buffer (NEB, #T2012) and following the Monarch kit (NEB, #T2010).

    Techniques: Binding Assay, Real-time Polymerase Chain Reaction, Negative Control, Positive Control

    Delivery of ACE-tRNA Arg as RNA to R1162X-CFTR 16HBEge cells results in significant rescue of CFTR mRNA expression (A) R1162X-CFTR cells stably expressing NLuc-PEST UGA reporter (R1162X PB PEST-NLuc UGA cells) were nucleofected with and without ACE-tRNA Arg RNA. Nonsense suppression mediated by ACE-tRNA Arg was detected by luminescence measurements at 2, 3, 4, 6, 8, 10, 12, 18, 24, and 30 h post-nucleofection. Based on the average decay phase of luminescence by subtracting luminescence measurement with no delivered tRNA from that with ACE-tRNA Arg RNA (red dotted line, y = 11,413 e −0.11x ), half-life of ACE-tRNA Arg is 6.5 ± 0.3 h. Raw luminescence measurements are presented in Figure S2 E. (B) After 6 h (left) of delivery, ACE-tRNA Arg (red filled) delivered as RNA in R1162X-CFTR cells is sufficient to significantly rescue R1162X-CFTR mRNA expression as determined by real-time qRT-PCR, from no RNA control (red open). No significant rescue of R1162X-CFTR mRNA expression was observed at 24 h (right). Data are presented as average ± SEM. All experiments contain an n = 3. Significance was determined by unpaired t test, where ∗∗p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Efficient suppression of endogenous CFTR nonsense mutations using anticodon-engineered transfer RNAs

    doi: 10.1016/j.omtn.2022.04.033

    Figure Lengend Snippet: Delivery of ACE-tRNA Arg as RNA to R1162X-CFTR 16HBEge cells results in significant rescue of CFTR mRNA expression (A) R1162X-CFTR cells stably expressing NLuc-PEST UGA reporter (R1162X PB PEST-NLuc UGA cells) were nucleofected with and without ACE-tRNA Arg RNA. Nonsense suppression mediated by ACE-tRNA Arg was detected by luminescence measurements at 2, 3, 4, 6, 8, 10, 12, 18, 24, and 30 h post-nucleofection. Based on the average decay phase of luminescence by subtracting luminescence measurement with no delivered tRNA from that with ACE-tRNA Arg RNA (red dotted line, y = 11,413 e −0.11x ), half-life of ACE-tRNA Arg is 6.5 ± 0.3 h. Raw luminescence measurements are presented in Figure S2 E. (B) After 6 h (left) of delivery, ACE-tRNA Arg (red filled) delivered as RNA in R1162X-CFTR cells is sufficient to significantly rescue R1162X-CFTR mRNA expression as determined by real-time qRT-PCR, from no RNA control (red open). No significant rescue of R1162X-CFTR mRNA expression was observed at 24 h (right). Data are presented as average ± SEM. All experiments contain an n = 3. Significance was determined by unpaired t test, where ∗∗p

    Article Snippet: Total RNA was isolated from cells with the Monarch Total RNA Miniprep Kit (NEB; no. T2010S) according to manufacturer’s recommendations.

    Techniques: Expressing, Stable Transfection, Quantitative RT-PCR