monarch total rna miniprep kit  (New England Biolabs)


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    Structured Review

    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 ∆QueC. Control (WT) and siEhDUF2419 trophozoites were cultivated in the presence of E. coli K12 or E. coli ∆QueC for 7 days (ration of 1 trophozoite:1000 bacteria). (1) WT trophozoites (2) queuine-treated WT trophozoites (3) WT trophozoites that were cultivated with E. coli K12 (4) WT trophozoites that were cultivated with E. coli ∆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 ∆QueC (9) E. coli K12 <t>RNA.</t> Data are from two biological replicates, each with two technical replicates. ** indicates p value
    Monarch Total Rna Miniprep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 98 stars, based on 26 article reviews
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    monarch total rna miniprep kit - by Bioz Stars, 2022-10
    98/100 stars

    Images

    1) Product Images from "Queuine Salvaging in the Human Parasite Entamoeba histolytica"

    Article Title: Queuine Salvaging in the Human Parasite Entamoeba histolytica

    Journal: Cells

    doi: 10.3390/cells11162509

    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 ∆QueC. Control (WT) and siEhDUF2419 trophozoites were cultivated in the presence of E. coli K12 or E. coli ∆QueC for 7 days (ration of 1 trophozoite:1000 bacteria). (1) WT trophozoites (2) queuine-treated WT trophozoites (3) WT trophozoites that were cultivated with E. coli K12 (4) WT trophozoites that were cultivated with E. coli ∆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 ∆QueC (9) E. coli K12 RNA. Data are from two biological replicates, each with two technical replicates. ** indicates p value
    Figure Legend 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 ∆QueC. Control (WT) and siEhDUF2419 trophozoites were cultivated in the presence of E. coli K12 or E. coli ∆QueC for 7 days (ration of 1 trophozoite:1000 bacteria). (1) WT trophozoites (2) queuine-treated WT trophozoites (3) WT trophozoites that were cultivated with E. coli K12 (4) WT trophozoites that were cultivated with E. coli ∆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 ∆QueC (9) E. coli K12 RNA. Data are from two biological replicates, each with two technical replicates. ** indicates p value

    Techniques Used: Northern Blot

    2) Product Images from "Queuine salvaging in the human parasite Entamoeba histolytica"

    Article Title: Queuine salvaging in the human parasite Entamoeba histolytica

    Journal: bioRxiv

    doi: 10.1101/2022.06.21.496972

    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
    Figure Legend 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

    Techniques Used: Northern Blot

    3) Product Images from "Bromodomain factor 5 is an essential regulator of transcription in Leishmania"

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

    Journal: Nature Communications

    doi: 10.1038/s41467-022-31742-1

    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.
    Figure Legend 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.

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

    4) Product Images from "Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function"

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

    Journal: bioRxiv

    doi: 10.1101/2022.05.20.492787

    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
    Figure Legend 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

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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).
    Figure Legend 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).

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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 .
    Figure Legend 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 .

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

    5) Product Images from "Single-tube isothermal label-free fluorescent sensor for pathogen detection based on genetic signatures"

    Article Title: Single-tube isothermal label-free fluorescent sensor for pathogen detection based on genetic signatures

    Journal: Frontiers in Chemistry

    doi: 10.3389/fchem.2022.951279

    Limit of detection and selectivity of the G4-MTC 3WJ system. (A) Dependence of the fluorescence intensity at 608 nm on MTC concentration upon incubation of the samples at 50°C for 30 min or 60 min. (B) Limit of detection for 30-min or 60-min assay. (C) Selectivity of the system in response to total bacterial RNA (17 ng/μL) from M. bovis BCG (MTC), M. simiae (Msim) or M. abscessus (Mabs). The signal is expressed as the fluorescence of the target-containing sample after subtraction of the no-target control. The data is averaged from three independent experiments, with standard deviations as error bars.
    Figure Legend Snippet: Limit of detection and selectivity of the G4-MTC 3WJ system. (A) Dependence of the fluorescence intensity at 608 nm on MTC concentration upon incubation of the samples at 50°C for 30 min or 60 min. (B) Limit of detection for 30-min or 60-min assay. (C) Selectivity of the system in response to total bacterial RNA (17 ng/μL) from M. bovis BCG (MTC), M. simiae (Msim) or M. abscessus (Mabs). The signal is expressed as the fluorescence of the target-containing sample after subtraction of the no-target control. The data is averaged from three independent experiments, with standard deviations as error bars.

    Techniques Used: Fluorescence, Concentration Assay, Incubation

    3WJ system expressing the NMM-binding G4 sequence in response to MTC. (A) . 3WJ complex formed between EP and TP strands in the presence of the MTC target. The dashed line connecting two fragments of the TP strand represents the hexaethylene glycol ( heg ) linker. The nucleotides corresponding to the promoter complement and the aptamer-encoding domain are in grey and green, respectively. (B) . Effect of a non-nucleotide linker on the fluorescent signal of the MTC-G4-3WJ system in the absence or presence of 100 nM MTC target. NTC stands for no-target control. The samples were incubated at 50°C for 30 min (C) . Analysis of the 3WJ complex and the product of its extension by Bsm DNA polymerase by native gel electrophoresis. All oligonucleotides were used at 100 nM. The samples were pre-incubated at 50°C for 30 min. (D) . Analysis of the G4 sequence expression in the presence of MTC (100 nM) by the system containing both Bsm DNA polymerase and T7 RNA polymerase using denaturing gel electrophoresis. (E) Fluorescent response of the system to MTC (10 or 100 nM). The bar corresponding to the no-target control is labeled as “0”. All samples were incubated at 50°C for 30 min. Inset: Visual observation of the signal in the sample upon excitation with a transilluminator. (F) Instantaneous monitoring of the signal triggered by 100 nM MTC target or background signal (NTC). For panels (B) , (E) and (F) , the data is average of three independent experiments, with error bars as standard deviations.
    Figure Legend Snippet: 3WJ system expressing the NMM-binding G4 sequence in response to MTC. (A) . 3WJ complex formed between EP and TP strands in the presence of the MTC target. The dashed line connecting two fragments of the TP strand represents the hexaethylene glycol ( heg ) linker. The nucleotides corresponding to the promoter complement and the aptamer-encoding domain are in grey and green, respectively. (B) . Effect of a non-nucleotide linker on the fluorescent signal of the MTC-G4-3WJ system in the absence or presence of 100 nM MTC target. NTC stands for no-target control. The samples were incubated at 50°C for 30 min (C) . Analysis of the 3WJ complex and the product of its extension by Bsm DNA polymerase by native gel electrophoresis. All oligonucleotides were used at 100 nM. The samples were pre-incubated at 50°C for 30 min. (D) . Analysis of the G4 sequence expression in the presence of MTC (100 nM) by the system containing both Bsm DNA polymerase and T7 RNA polymerase using denaturing gel electrophoresis. (E) Fluorescent response of the system to MTC (10 or 100 nM). The bar corresponding to the no-target control is labeled as “0”. All samples were incubated at 50°C for 30 min. Inset: Visual observation of the signal in the sample upon excitation with a transilluminator. (F) Instantaneous monitoring of the signal triggered by 100 nM MTC target or background signal (NTC). For panels (B) , (E) and (F) , the data is average of three independent experiments, with error bars as standard deviations.

    Techniques Used: Expressing, Binding Assay, Sequencing, Incubation, Nucleic Acid Electrophoresis, Labeling

    Performance of the 3WJ system expressing the G4-folding sequence in response to a fragment of E. coli 16S rRNA. (A) 3WJ complex that presumably forms between EP and TP strands in the presence of the EC target. The dashed line connecting two fragments of the TP strand represents the heg linker. The nucleotides corresponding to the promoter complement and the aptamer-encoding domain are in grey and green, respectively. (B) Analysis of the 3WJ complex and the product of its extension by Bsm DNA polymerase (ext-3WJ) by native polyacrylamide gel electrophoresis. All oligonucleotides were used at 100 nM. The samples were pre-incubated at 50°C for 30 min. (C) Fluorescent response of the system to the absence of targets (NTC, “no-target control”) or to the presence of either synthetic target EC (20 nM) or total E. coli RNA preparation containing 30 ng/ul of 16S rRNA. The samples were incubated at 50°C for 30 min. The data is averaged from three independent experiments, with error bars as standard deviations. The dashed line shows the fluorescent intensity threshold corresponding to the average intensity of the NTC sample plus 3 standard deviations. The signal above the threshold indicates the presence of the interrogated target.
    Figure Legend Snippet: Performance of the 3WJ system expressing the G4-folding sequence in response to a fragment of E. coli 16S rRNA. (A) 3WJ complex that presumably forms between EP and TP strands in the presence of the EC target. The dashed line connecting two fragments of the TP strand represents the heg linker. The nucleotides corresponding to the promoter complement and the aptamer-encoding domain are in grey and green, respectively. (B) Analysis of the 3WJ complex and the product of its extension by Bsm DNA polymerase (ext-3WJ) by native polyacrylamide gel electrophoresis. All oligonucleotides were used at 100 nM. The samples were pre-incubated at 50°C for 30 min. (C) Fluorescent response of the system to the absence of targets (NTC, “no-target control”) or to the presence of either synthetic target EC (20 nM) or total E. coli RNA preparation containing 30 ng/ul of 16S rRNA. The samples were incubated at 50°C for 30 min. The data is averaged from three independent experiments, with error bars as standard deviations. The dashed line shows the fluorescent intensity threshold corresponding to the average intensity of the NTC sample plus 3 standard deviations. The signal above the threshold indicates the presence of the interrogated target.

    Techniques Used: Expressing, Sequencing, Polyacrylamide Gel Electrophoresis, Incubation

    Real-time light-up aptamer-expressing 3WJ system. The signal is due to the following steps occurring in the same sample: (1) An RNA target is interrogated by the template probe (TP) and extension probe (EP) to form a 3WJ structure; (2) the 3’-end of EP in the 3WJ structure is extended to form T7 RNA polymerase promoter sequence and the template encoding the sequence of a light-up aptamer, which is complementary to the “aptamer template” fragment of TP; (3) the RNA polymerase recognizes the promotor sequence and generates multiple copies of the encoded RNA aptamer, which acquires the active dye-binding conformation to enhance fluorescence of a cognate fluorogenic dye (4).
    Figure Legend Snippet: Real-time light-up aptamer-expressing 3WJ system. The signal is due to the following steps occurring in the same sample: (1) An RNA target is interrogated by the template probe (TP) and extension probe (EP) to form a 3WJ structure; (2) the 3’-end of EP in the 3WJ structure is extended to form T7 RNA polymerase promoter sequence and the template encoding the sequence of a light-up aptamer, which is complementary to the “aptamer template” fragment of TP; (3) the RNA polymerase recognizes the promotor sequence and generates multiple copies of the encoded RNA aptamer, which acquires the active dye-binding conformation to enhance fluorescence of a cognate fluorogenic dye (4).

    Techniques Used: Expressing, Sequencing, Binding Assay, Fluorescence

    6) Product Images from "Efficient suppression of endogenous CFTR nonsense mutations using anticodon-engineered transfer RNAs"

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

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2022.04.033

    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
    Figure Legend 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

    Techniques Used: Expressing, Stable Transfection, Quantitative RT-PCR

    7) Product Images from "Nicked tRNAs are stable reservoirs of tRNA halves in cells and biofluids"

    Article Title: Nicked tRNAs are stable reservoirs of tRNA halves in cells and biofluids

    Journal: bioRxiv

    doi: 10.1101/2022.08.31.506125

    Nonvesicular tRNA halves circulating in human biofluids and intracellular glycine tiRNAs are predominantly nicked tRNAs. A) Purified RNA from arsenite-treated U2-OS cells was separated on a Superdex 75 column using an FPLC system. Inset: northern blot of intracellular RNAs showing the presence of tRNA halves in the input. Selected fractions from (A) were analyzed by northern blot (B) or by stem-loop RT-qPCR (C). C q values were normalized to the fraction containing the highest signal. A-C bottom panels: the RNA was heat-denatured before injection. D) Cells were transfected with synthetic 5’ tRNA Gly [9 GG/AA ], then lysed with SDS as described in ( Tosar et al., 2018 ). The lysate was separated by SEC and fractions analyzed by SL-RT-qPCR using primers specific for the 9 GG/AA sequence. E–F) Separation by SEC of purified RNA from Proteinase K-treated ultracentrifugation supernatants of human serum (E) or CSF (F). Selected eluted fractions were analyzed by SL-RT-qPCR using primers specific for 5’ tRNA Gly GCC halves of 30 nt (red) and miR-21-5p (green). For reference, a tRNA icon in this figure indicates fractions where full-length tRNAs are expected to elute (if present).
    Figure Legend Snippet: Nonvesicular tRNA halves circulating in human biofluids and intracellular glycine tiRNAs are predominantly nicked tRNAs. A) Purified RNA from arsenite-treated U2-OS cells was separated on a Superdex 75 column using an FPLC system. Inset: northern blot of intracellular RNAs showing the presence of tRNA halves in the input. Selected fractions from (A) were analyzed by northern blot (B) or by stem-loop RT-qPCR (C). C q values were normalized to the fraction containing the highest signal. A-C bottom panels: the RNA was heat-denatured before injection. D) Cells were transfected with synthetic 5’ tRNA Gly [9 GG/AA ], then lysed with SDS as described in ( Tosar et al., 2018 ). The lysate was separated by SEC and fractions analyzed by SL-RT-qPCR using primers specific for the 9 GG/AA sequence. E–F) Separation by SEC of purified RNA from Proteinase K-treated ultracentrifugation supernatants of human serum (E) or CSF (F). Selected eluted fractions were analyzed by SL-RT-qPCR using primers specific for 5’ tRNA Gly GCC halves of 30 nt (red) and miR-21-5p (green). For reference, a tRNA icon in this figure indicates fractions where full-length tRNAs are expected to elute (if present).

    Techniques Used: Purification, Fast Protein Liquid Chromatography, Northern Blot, Quantitative RT-PCR, Injection, Transfection, Sequencing

    Identification of stable nonvesicular RNAs. Northern blot of different rRNAs and tRNAs after incubating purified RNA (A) or ribosomes (B) from human cells in 10% FBS. C-D) Northern blot of 5’ tRNA Gly GCC (D, left) or 5’ 28S rRNA-derived fragments (D, right) in extracellular nonvesicular fractions purified by density gradients (C). U2-OS cells were treated or not with NaAsO 2 (ARS) before collecting the cell-conditioned medium (CCM). E) Read coverage in small RNA-seq data of extracellular ribosomes (from Tosar et al., 2020 ), revealing enrichment of 40 nt 5’-derived small RNAs among all other 28S rRNA-derived fragments.
    Figure Legend Snippet: Identification of stable nonvesicular RNAs. Northern blot of different rRNAs and tRNAs after incubating purified RNA (A) or ribosomes (B) from human cells in 10% FBS. C-D) Northern blot of 5’ tRNA Gly GCC (D, left) or 5’ 28S rRNA-derived fragments (D, right) in extracellular nonvesicular fractions purified by density gradients (C). U2-OS cells were treated or not with NaAsO 2 (ARS) before collecting the cell-conditioned medium (CCM). E) Read coverage in small RNA-seq data of extracellular ribosomes (from Tosar et al., 2020 ), revealing enrichment of 40 nt 5’-derived small RNAs among all other 28S rRNA-derived fragments.

    Techniques Used: Northern Blot, Purification, Derivative Assay, RNA Sequencing Assay

    Nicked tRNAs protect tRNA halves from degradation, are dissociated by phenol, and can be repaired by RtcB. RNA purification by the miRNeasy micro kit (A) or TRIzol (TRI, B) impairs enzymatic (PNK+Rnl1) nicked tRNA repair (blue arrows). C) Purification of RNase1-treated RNA by SPE and re-exposure to RNase 1, with or without previous heat denaturation. D) Northern blot of RNAse1-treated RNA purified by SPE, TRIzol, miRNeasy or heated before SPE purification, after separation in native gels. E) Nicked tRNA repair with RtCB from E. coli .
    Figure Legend Snippet: Nicked tRNAs protect tRNA halves from degradation, are dissociated by phenol, and can be repaired by RtcB. RNA purification by the miRNeasy micro kit (A) or TRIzol (TRI, B) impairs enzymatic (PNK+Rnl1) nicked tRNA repair (blue arrows). C) Purification of RNase1-treated RNA by SPE and re-exposure to RNase 1, with or without previous heat denaturation. D) Northern blot of RNAse1-treated RNA purified by SPE, TRIzol, miRNeasy or heated before SPE purification, after separation in native gels. E) Nicked tRNA repair with RtCB from E. coli .

    Techniques Used: Purification, Northern Blot

    Most tRNA halves identified by northern blot are nicked tRNAs. A) RNase 1-treated RNA (60 min) was incubated with the indicated enzymatic combinations. Repair of tRNA Gly GCC was analyzed by northern blot using a 5’-targeting probe. B) schematic representation of the nicked tRNA repair strategy and 5’ (blue) and 3’ (orange) probe binding sites in tRNA Gly GCC . C) Design of a third probe targeting the anticodon loop (ACL) of tRNA Gly GCC and tRNA Asp GUC . D) enzymatic repair of tRNA Gly GCC evidenced with either the 5’, ACL or 3’ probes. NT: fragmented RNA purified by SPE but without treatment with the enzymatic repair cocktail. (-)PNK: mutant PNK lacking phosphatase activity. Δ: heat.
    Figure Legend Snippet: Most tRNA halves identified by northern blot are nicked tRNAs. A) RNase 1-treated RNA (60 min) was incubated with the indicated enzymatic combinations. Repair of tRNA Gly GCC was analyzed by northern blot using a 5’-targeting probe. B) schematic representation of the nicked tRNA repair strategy and 5’ (blue) and 3’ (orange) probe binding sites in tRNA Gly GCC . C) Design of a third probe targeting the anticodon loop (ACL) of tRNA Gly GCC and tRNA Asp GUC . D) enzymatic repair of tRNA Gly GCC evidenced with either the 5’, ACL or 3’ probes. NT: fragmented RNA purified by SPE but without treatment with the enzymatic repair cocktail. (-)PNK: mutant PNK lacking phosphatase activity. Δ: heat.

    Techniques Used: Northern Blot, Incubation, Binding Assay, Purification, Mutagenesis, Activity Assay

    Naked tRNA halves are extremely stable in human biofluids. Northern blot of several noncoding transcripts after incubating purified RNA from human cells in 10% FBS or with recombinant human RNase 1 for different periods (A). Samples were also incubated in human urine, 10% serum and CSF (B). Half-lives in CSF were calculated for all tested RNAs and shown in (C). r-RNase1: recombinant human RNase 1.
    Figure Legend Snippet: Naked tRNA halves are extremely stable in human biofluids. Northern blot of several noncoding transcripts after incubating purified RNA from human cells in 10% FBS or with recombinant human RNase 1 for different periods (A). Samples were also incubated in human urine, 10% serum and CSF (B). Half-lives in CSF were calculated for all tested RNAs and shown in (C). r-RNase1: recombinant human RNase 1.

    Techniques Used: Northern Blot, Purification, Recombinant, Incubation

    8) Product Images from "A versatile active learning workflow for optimization of genetic and metabolic networks"

    Article Title: A versatile active learning workflow for optimization of genetic and metabolic networks

    Journal: Nature Communications

    doi: 10.1038/s41467-022-31245-z

    Application of METIS for optimization of a transcription translation unit. a The cell-free expression of sfGfp (super-folder Gfp ) using plasmid, linear DNA (PCR) and linear DNA plus GamS protein, a nuclease inhibitor that protects linear DNA from degradation. The bars and the error bars are the average and standard deviation of triplicates ( n = 3 independent experiments), respectively. b Design of a transcription translation unit controlled by variants of a T7 promoter, ribosome binding site (RBS), N-terminal amino acids 3, 4, and 5, and the last two C-terminal amino acids. The combinatorial transcription translation units are expressed from linear DNA in the TXTL system consisting of the E. coli lysate, buffer and energy mix, as well as purified GamS and T7 RNA polymerase. c The plot representing the average of triplicates ( n = 3 independent experiments) as the result of 4 rounds of active learning, with 50 transcription translation units tested per round. The yield is the Gfp fluorescence readout after 6 hours at 30 °C normalized by the same value from the reference constructs commonly used in the lab (Methods). The gray lines show the median. d A list of 20 most informative combinations of 4-day active learning performed in the cell-free system (c) was downloaded and the combinations were cloned in a vector and transformed into E. coli DH10β harboring a plasmid expressing auto-regulated T7 RNA polymerase (Methods). e Cell-free versus in vivo yields (average and standard deviation of triplicates, n = 3 independent experiments) for the 20 most informative combinations. f In vivo yield results (average of triplicates, n = 3 independent experiments) of Day 0 (20 most informative combinations) and Day 1 (suggested by the workflow). The gray lines show the median. The Google Colab Python notebook and all active learning data (combinations and yields) in this figure are available at https://github.com/amirpandi/METIS . Source data for a , e are provided as a Source Data file.
    Figure Legend Snippet: Application of METIS for optimization of a transcription translation unit. a The cell-free expression of sfGfp (super-folder Gfp ) using plasmid, linear DNA (PCR) and linear DNA plus GamS protein, a nuclease inhibitor that protects linear DNA from degradation. The bars and the error bars are the average and standard deviation of triplicates ( n = 3 independent experiments), respectively. b Design of a transcription translation unit controlled by variants of a T7 promoter, ribosome binding site (RBS), N-terminal amino acids 3, 4, and 5, and the last two C-terminal amino acids. The combinatorial transcription translation units are expressed from linear DNA in the TXTL system consisting of the E. coli lysate, buffer and energy mix, as well as purified GamS and T7 RNA polymerase. c The plot representing the average of triplicates ( n = 3 independent experiments) as the result of 4 rounds of active learning, with 50 transcription translation units tested per round. The yield is the Gfp fluorescence readout after 6 hours at 30 °C normalized by the same value from the reference constructs commonly used in the lab (Methods). The gray lines show the median. d A list of 20 most informative combinations of 4-day active learning performed in the cell-free system (c) was downloaded and the combinations were cloned in a vector and transformed into E. coli DH10β harboring a plasmid expressing auto-regulated T7 RNA polymerase (Methods). e Cell-free versus in vivo yields (average and standard deviation of triplicates, n = 3 independent experiments) for the 20 most informative combinations. f In vivo yield results (average of triplicates, n = 3 independent experiments) of Day 0 (20 most informative combinations) and Day 1 (suggested by the workflow). The gray lines show the median. The Google Colab Python notebook and all active learning data (combinations and yields) in this figure are available at https://github.com/amirpandi/METIS . Source data for a , e are provided as a Source Data file.

    Techniques Used: Expressing, Plasmid Preparation, Polymerase Chain Reaction, Standard Deviation, Binding Assay, Purification, Fluorescence, Construct, Clone Assay, Transformation Assay, In Vivo

    Application of METIS for optimization of a LacI gene circuit. a LacI gene circuits characterized by dynamic range (DR) and fold-change (FC) of the output (Gfp fluorescence) between 0 and 10 mM IPTG. b Active learning by varying components of E. coli TXTL, 4 lacI circuit plasmids as alternatives, T7 RNA polymerase and a T7- lacI plasmid. c The objective function (FC × DR) and fold change (FC) values, average of triplicates ( n = 3 independent experiments) in 10 rounds of active learning. The gray lines show the median. d The distribution yield values within the range of each factor. e Feature importance percentages showing the effect of each factor on the objective function. f Titration of P T7 - LacI plasmid and T7 RNA polymerase with the optimal composition (from active learning that achieved with pTHS circuit). The heatmaps show FC × DR (left) and FC (right) values (average of triplicates, n = 3 independent experiments) of the titration. g Fluorescence values (average of triplicates, n = 3 independent experiments) of the similar titration as in f but instead of the pTHS circuit, a Gfp expressing plasmid was used). h Titration of LacI plasmids with constitutive/T7 promoter in combination with a Gfp plasmid with constitutive/T7 promoter. i The RT-qPCR results of the relative level of LacI and Gfp mRNAs after 10 h. Relative log2 resource share between LacI and Gfp mRNA in each sample is reported to account for RNA purification efficiency variability. In h and i bars are the average of triplicates ( n = 3 independent experiments) and error bars are standard deviation. j Usage of the METIS module, K most informative combinations for further LacI circuit optimization. k Objective function FC × DR and FC (average of triplicates, n = 3 independent experiments) of 20 most informative combinations with purified LacI (Day 0) followed by Day 1 experiments suggested by METIS. The gray lines show the median. The Google Colab Python notebook and all active learning data (combinations and yields) in this figure are available at https://github.com/amirpandi/METIS . Source data for f – i are provided as a Source Data file.
    Figure Legend Snippet: Application of METIS for optimization of a LacI gene circuit. a LacI gene circuits characterized by dynamic range (DR) and fold-change (FC) of the output (Gfp fluorescence) between 0 and 10 mM IPTG. b Active learning by varying components of E. coli TXTL, 4 lacI circuit plasmids as alternatives, T7 RNA polymerase and a T7- lacI plasmid. c The objective function (FC × DR) and fold change (FC) values, average of triplicates ( n = 3 independent experiments) in 10 rounds of active learning. The gray lines show the median. d The distribution yield values within the range of each factor. e Feature importance percentages showing the effect of each factor on the objective function. f Titration of P T7 - LacI plasmid and T7 RNA polymerase with the optimal composition (from active learning that achieved with pTHS circuit). The heatmaps show FC × DR (left) and FC (right) values (average of triplicates, n = 3 independent experiments) of the titration. g Fluorescence values (average of triplicates, n = 3 independent experiments) of the similar titration as in f but instead of the pTHS circuit, a Gfp expressing plasmid was used). h Titration of LacI plasmids with constitutive/T7 promoter in combination with a Gfp plasmid with constitutive/T7 promoter. i The RT-qPCR results of the relative level of LacI and Gfp mRNAs after 10 h. Relative log2 resource share between LacI and Gfp mRNA in each sample is reported to account for RNA purification efficiency variability. In h and i bars are the average of triplicates ( n = 3 independent experiments) and error bars are standard deviation. j Usage of the METIS module, K most informative combinations for further LacI circuit optimization. k Objective function FC × DR and FC (average of triplicates, n = 3 independent experiments) of 20 most informative combinations with purified LacI (Day 0) followed by Day 1 experiments suggested by METIS. The gray lines show the median. The Google Colab Python notebook and all active learning data (combinations and yields) in this figure are available at https://github.com/amirpandi/METIS . Source data for f – i are provided as a Source Data file.

    Techniques Used: Fluorescence, Plasmid Preparation, Titration, Expressing, Quantitative RT-PCR, Purification, Standard Deviation

    9) Product Images from "Engineered model of t(7;12)(q36;p13) AML recapitulates patient-specific features and gene expression profiles"

    Article Title: Engineered model of t(7;12)(q36;p13) AML recapitulates patient-specific features and gene expression profiles

    Journal: bioRxiv

    doi: 10.1101/2022.06.14.496084

    Comparison of K562-t(7;12) transcriptional landscape with t(7;12) patient signatures. A) 177-gene signature of t(7;12)-patient gene expression extrapolated from published microarray and RNA sequencing datasets by comparison with other paediatric AML subtypes. The Venn diagram shows the 177 intersect genes. B) 121-gene t(7;12)-specific signature inferred by comparisons of gene expressions of paediatric AML and normal bone marrow (NBM) samples. Edward’s Venn diagram highlights the 121 exclusive genes to t(7;12). C-D) Gene Ontology analysis of the 177-signature and 121-signature by the PANTHER annotation repository of biological processes (BP). E-F) GSEA enrichment plots of K562-t(7;12) gene expression profile using the 177- and 121-signatures. NES = normalized enrichment score; FDR = false discovery rate. G) Core enriched genes from the GSEA using the 177- and 121-signatures, shown by their enrichment index in K562-t(7;12) against K562 control. MNX1 is highlighted by the arrows.
    Figure Legend Snippet: Comparison of K562-t(7;12) transcriptional landscape with t(7;12) patient signatures. A) 177-gene signature of t(7;12)-patient gene expression extrapolated from published microarray and RNA sequencing datasets by comparison with other paediatric AML subtypes. The Venn diagram shows the 177 intersect genes. B) 121-gene t(7;12)-specific signature inferred by comparisons of gene expressions of paediatric AML and normal bone marrow (NBM) samples. Edward’s Venn diagram highlights the 121 exclusive genes to t(7;12). C-D) Gene Ontology analysis of the 177-signature and 121-signature by the PANTHER annotation repository of biological processes (BP). E-F) GSEA enrichment plots of K562-t(7;12) gene expression profile using the 177- and 121-signatures. NES = normalized enrichment score; FDR = false discovery rate. G) Core enriched genes from the GSEA using the 177- and 121-signatures, shown by their enrichment index in K562-t(7;12) against K562 control. MNX1 is highlighted by the arrows.

    Techniques Used: Expressing, Microarray, RNA Sequencing Assay

    Differentially expressed haemoglobin genes in K562-t(7;12). Gene expressions were extracted from RNA sequencing counts of K562-t(7;12) and K562 control. Blue shading indicates a significant downregulation determined by T-test. Asterisks symbolise p value thresholds of 0.05 (*), 0.001 (**), 0.0001 (***), and 0.00001 (****).
    Figure Legend Snippet: Differentially expressed haemoglobin genes in K562-t(7;12). Gene expressions were extracted from RNA sequencing counts of K562-t(7;12) and K562 control. Blue shading indicates a significant downregulation determined by T-test. Asterisks symbolise p value thresholds of 0.05 (*), 0.001 (**), 0.0001 (***), and 0.00001 (****).

    Techniques Used: RNA Sequencing Assay

    Expression differences in K562-t(7;12) compared to K562 control of known t(7;12)-associated genes. Gene expressions were extracted from RNA sequencing counts, and grouped by gene families. Red squares around the plot indicate a gene previously reported by Wildenhain et al . (2010) or Balgobind et al . (2011) as specific for the t(7;12) subtype. Grey shading indicates that the gene was not significantly dysregulated in K562-t(7;12) determined by T-test, while red indicates a significant upregulation, and blue a significant downregulation. Asterisks symbolise p value thresholds of 0.05 (*), 0.001 (**), 0.0001 (***), and 0.00001 (****).
    Figure Legend Snippet: Expression differences in K562-t(7;12) compared to K562 control of known t(7;12)-associated genes. Gene expressions were extracted from RNA sequencing counts, and grouped by gene families. Red squares around the plot indicate a gene previously reported by Wildenhain et al . (2010) or Balgobind et al . (2011) as specific for the t(7;12) subtype. Grey shading indicates that the gene was not significantly dysregulated in K562-t(7;12) determined by T-test, while red indicates a significant upregulation, and blue a significant downregulation. Asterisks symbolise p value thresholds of 0.05 (*), 0.001 (**), 0.0001 (***), and 0.00001 (****).

    Techniques Used: Expressing, RNA Sequencing Assay

    Generation of t(7;12)(q36;p13) in K562 cells. A) Schematic representation of target regions for CRISPR/Cas9-directed cleavage on chromosomes 7 and 12 used for guide RNA (gRNA) design. Fusion junctions are flanked by arrows representing PCR primers used for confirmation, yielding products of 614 bp and 937 bp. Below, detailed location of the targeted regions on chromosome 7q36.3 and 12p13.2 with reference to known t(7;12) breakpoints used to generate the translocation. B) FISH using the specific t(7;12) probe XL t(7;12) MNX1/ETV6 (MetaSystems Gmbh, Altlussheim, Germany, Supplementary Table 3 ) hybridising chromosome 7q36 in red and chromosome 12p13 in green. Two yellow fusion signals, pointed by arrows, indicate the presence of t(7;12) in a representative interphase nucleus of K562-t(7;12). The karyotype of K562 is nearly tetraploid and harbours complex rearrangements, including a duplication of the 7q36 locus within the short arm of chromosome 7, hence showing five 7q36 signals and two 12p13 signals ( 30 ) ( Supplementary Figure 2 ). C) Metaphase spread of K562-t(7;12) hybridised with the same probe shows the presence of derivative chromosomes der( 7 ) and der( 12 ), pointed by arrows. D) Confirmation of the presence of t(7;12) in K562-t(7;12) but not K562 control (‘ctrl’) cells (K562 electroporated with Cas9 only) by PCR amplification of fusion junctions and product separation on agarose gel; bands correspond to the predicted sizes shown in panel A. NTC = no template control. E) qRT-PCR validation of overexpression of MNX1 in K562-t(7;12) compared to K562 control. The fold change was calculated using the ΔΔCt method by normalisation to the endogenous gene HPRT1 . Error bars represent standard deviation (SD) of n=3. Primers are reported in Supplementary Table 4. F ) A custom-made 3-colour probe (MetaSystems dual-colour ETV6 + PAC-derived RP5-1121A15) hybridising ETV6 portions in red (centromeric) and green (telomeric), and the MNX1 locus in cyan ( Supplementary Figure 2 ; Supplementary Table 3 ), allowed visualisation of both derivative chromosomes in interphase nuclei (pointed by yellow arrows). The white radius arrow represents the distance between nuclear interior (value=0) and nuclear periphery (value=1), which was used in the calculation of radial nuclear locations (RNL). G) RNL of der( 7 ) and der( 12 ) signals in K562-t(7;12) interphase nuclei. The RNL values are expressed as median values of individual localisations of FISH signals (200 nuclei analysed per condition). Errors bars represent standard error of the mean (SEM). Values closer to 0 indicate an internal position within the nucleus (described in detail in Federico et al . ( 31 )).
    Figure Legend Snippet: Generation of t(7;12)(q36;p13) in K562 cells. A) Schematic representation of target regions for CRISPR/Cas9-directed cleavage on chromosomes 7 and 12 used for guide RNA (gRNA) design. Fusion junctions are flanked by arrows representing PCR primers used for confirmation, yielding products of 614 bp and 937 bp. Below, detailed location of the targeted regions on chromosome 7q36.3 and 12p13.2 with reference to known t(7;12) breakpoints used to generate the translocation. B) FISH using the specific t(7;12) probe XL t(7;12) MNX1/ETV6 (MetaSystems Gmbh, Altlussheim, Germany, Supplementary Table 3 ) hybridising chromosome 7q36 in red and chromosome 12p13 in green. Two yellow fusion signals, pointed by arrows, indicate the presence of t(7;12) in a representative interphase nucleus of K562-t(7;12). The karyotype of K562 is nearly tetraploid and harbours complex rearrangements, including a duplication of the 7q36 locus within the short arm of chromosome 7, hence showing five 7q36 signals and two 12p13 signals ( 30 ) ( Supplementary Figure 2 ). C) Metaphase spread of K562-t(7;12) hybridised with the same probe shows the presence of derivative chromosomes der( 7 ) and der( 12 ), pointed by arrows. D) Confirmation of the presence of t(7;12) in K562-t(7;12) but not K562 control (‘ctrl’) cells (K562 electroporated with Cas9 only) by PCR amplification of fusion junctions and product separation on agarose gel; bands correspond to the predicted sizes shown in panel A. NTC = no template control. E) qRT-PCR validation of overexpression of MNX1 in K562-t(7;12) compared to K562 control. The fold change was calculated using the ΔΔCt method by normalisation to the endogenous gene HPRT1 . Error bars represent standard deviation (SD) of n=3. Primers are reported in Supplementary Table 4. F ) A custom-made 3-colour probe (MetaSystems dual-colour ETV6 + PAC-derived RP5-1121A15) hybridising ETV6 portions in red (centromeric) and green (telomeric), and the MNX1 locus in cyan ( Supplementary Figure 2 ; Supplementary Table 3 ), allowed visualisation of both derivative chromosomes in interphase nuclei (pointed by yellow arrows). The white radius arrow represents the distance between nuclear interior (value=0) and nuclear periphery (value=1), which was used in the calculation of radial nuclear locations (RNL). G) RNL of der( 7 ) and der( 12 ) signals in K562-t(7;12) interphase nuclei. The RNL values are expressed as median values of individual localisations of FISH signals (200 nuclei analysed per condition). Errors bars represent standard error of the mean (SEM). Values closer to 0 indicate an internal position within the nucleus (described in detail in Federico et al . ( 31 )).

    Techniques Used: CRISPR, Polymerase Chain Reaction, Translocation Assay, Fluorescence In Situ Hybridization, Amplification, Agarose Gel Electrophoresis, Quantitative RT-PCR, Over Expression, Standard Deviation, Derivative Assay

    10) Product Images from "A versatile active learning workflow for optimization of genetic and metabolic networks"

    Article Title: A versatile active learning workflow for optimization of genetic and metabolic networks

    Journal: bioRxiv

    doi: 10.1101/2021.12.28.474323

    Application of METIS for optimization of a LacI gene circuit. a) Single and multi-level controller LacI gene circuits from Greco et al . 43 Characterization of these circuits through dynamic range (DR) and/or fold-change (FC) of the output (Gfp fluorescence) between 0 and 10 mM input (concentration of IPTG). b) Imported in the active learning notebook, the varied components of the reactions included 4 lacI circuits as alternatives, some factors of buffer and energy mix of E. coli cell-free system along with the lysate, as well as T7 RNA polymerase and a second plasmid expressing lacI under a T7 promoter. c) The average of triplicates as the result of 10 rounds of active learning as plots for the objective function (FC × DR) and fold change (FC) values. d) Plots showing the distribution of measured yield values within the ranges of each factor. e) Feature importance percentages showing the effect of each factor on decision-making by the model to predict objective function values. f) Titration of P T7 - LacI plasmid and T7 RNA polymerase with the optimal composition (from 10 rounds of active learning that achieved with pTHS LacI circuit, the toehold switch as the second level controller of gene expression through translation). The heatmaps show FC × DR (left) and FC (right) values (average of triplicates) of the titration. g) The same titration experiment as in (f) but instead of the pTHS circuit, Gfp was expressed under a constitutive promoter (independent from the P T7 - LacI plasmid and T7 RNA polymerase on the protein production). h) Titration (0, 1, 3, 10, 30, and 100 nM) of LacI plasmids with either constitutive or T7 promoter in combination with 10 nM of a Gfp plasmid (with T7 or constitutive promoter). i) The RT-qPCR results of the relative level of LacI and Gfp mRNAs for a similar experiment in (h) (0, 10, 100 nM LacI plasmids, and 10 nM Gfp plasmids) after 10 hours. Relative log2 resource share between LacI and Gfp mRNA in each sample is reported in order to account for RNA purification efficiency variability j) The 20 most informative combinations were downloaded after the 10-round active learning and the P T7 - LacI plasmid with purified LacI were replaced. After performing the experiments and measuring the objective function, we imported them as Day 0 and continued with experiments of the next round’s predictions (Day 1). k) Plots of the objective function FC × DR (left) and FC (right) values (average of triplicates) of 20 most informative combinations with purified LacI followed by Day 1 experiments suggested by the workflow. See Data availability for combinations and objective function values.
    Figure Legend Snippet: Application of METIS for optimization of a LacI gene circuit. a) Single and multi-level controller LacI gene circuits from Greco et al . 43 Characterization of these circuits through dynamic range (DR) and/or fold-change (FC) of the output (Gfp fluorescence) between 0 and 10 mM input (concentration of IPTG). b) Imported in the active learning notebook, the varied components of the reactions included 4 lacI circuits as alternatives, some factors of buffer and energy mix of E. coli cell-free system along with the lysate, as well as T7 RNA polymerase and a second plasmid expressing lacI under a T7 promoter. c) The average of triplicates as the result of 10 rounds of active learning as plots for the objective function (FC × DR) and fold change (FC) values. d) Plots showing the distribution of measured yield values within the ranges of each factor. e) Feature importance percentages showing the effect of each factor on decision-making by the model to predict objective function values. f) Titration of P T7 - LacI plasmid and T7 RNA polymerase with the optimal composition (from 10 rounds of active learning that achieved with pTHS LacI circuit, the toehold switch as the second level controller of gene expression through translation). The heatmaps show FC × DR (left) and FC (right) values (average of triplicates) of the titration. g) The same titration experiment as in (f) but instead of the pTHS circuit, Gfp was expressed under a constitutive promoter (independent from the P T7 - LacI plasmid and T7 RNA polymerase on the protein production). h) Titration (0, 1, 3, 10, 30, and 100 nM) of LacI plasmids with either constitutive or T7 promoter in combination with 10 nM of a Gfp plasmid (with T7 or constitutive promoter). i) The RT-qPCR results of the relative level of LacI and Gfp mRNAs for a similar experiment in (h) (0, 10, 100 nM LacI plasmids, and 10 nM Gfp plasmids) after 10 hours. Relative log2 resource share between LacI and Gfp mRNA in each sample is reported in order to account for RNA purification efficiency variability j) The 20 most informative combinations were downloaded after the 10-round active learning and the P T7 - LacI plasmid with purified LacI were replaced. After performing the experiments and measuring the objective function, we imported them as Day 0 and continued with experiments of the next round’s predictions (Day 1). k) Plots of the objective function FC × DR (left) and FC (right) values (average of triplicates) of 20 most informative combinations with purified LacI followed by Day 1 experiments suggested by the workflow. See Data availability for combinations and objective function values.

    Techniques Used: Fluorescence, Concentration Assay, Plasmid Preparation, Expressing, Titration, Quantitative RT-PCR, Purification

    11) Product Images from "Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function"

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

    Journal: bioRxiv

    doi: 10.1101/2022.05.20.492787

    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
    Figure Legend 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

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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).
    Figure Legend 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).

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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 .
    Figure Legend 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 .

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

    12) Product Images from "Bromodomain factor 5 is an essential regulator of transcription in Leishmania"

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

    Journal: Nature Communications

    doi: 10.1038/s41467-022-31742-1

    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.
    Figure Legend 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.

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

    13) Product Images from "Bromodomain factor 5 is an essential transcriptional regulator of the Leishmania genome"

    Article Title: Bromodomain factor 5 is an essential transcriptional regulator of the Leishmania genome

    Journal: bioRxiv

    doi: 10.1101/2021.09.29.462384

    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. N=3 C. Metaplot divergent SSR (n=60) for DMSO treated or rapamycin-treated BDF5 −/+ flx . 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.
    Figure Legend 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. N=3 C. Metaplot divergent SSR (n=60) for DMSO treated or rapamycin-treated BDF5 −/+ flx . 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.

    Techniques Used: Expressing, Flow Cytometry, Staining, RNA Sequencing Assay

    14) Product Images from "Bromodomain factor 5 is an essential transcriptional regulator of the Leishmania genome"

    Article Title: Bromodomain factor 5 is an essential transcriptional regulator of the Leishmania genome

    Journal: bioRxiv

    doi: 10.1101/2021.09.29.462384

    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. N=3 C. Metaplot divergent SSR (n=60) for DMSO treated or rapamycin-treated BDF5 −/+ flx . 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.
    Figure Legend 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. N=3 C. Metaplot divergent SSR (n=60) for DMSO treated or rapamycin-treated BDF5 −/+ flx . 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.

    Techniques Used: Expressing, Flow Cytometry, Staining, RNA Sequencing Assay

    15) Product Images from "Bromodomain factor 5 is an essential regulator of transcription in Leishmania"

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

    Journal: Nature Communications

    doi: 10.1038/s41467-022-31742-1

    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.
    Figure Legend 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.

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

    16) Product Images from "Targeting Nup358/RanBP2 by a viral protein disrupts stress granule formation"

    Article Title: Targeting Nup358/RanBP2 by a viral protein disrupts stress granule formation

    Journal: bioRxiv

    doi: 10.1101/2022.05.19.492599

    CrPV-1A localizes to the nucleus and induces poly(A)+ RNA accumulation in the nucleus. (A) Confocal immunofluorescence images of S2 cells transfected with in vitro transcribed RNA encoding CrPV-1A, CrPV-1A(R146A), or CrPV-1A(F114A) for 16 hours. GFP fluorescence (green), CrPV-1A antibody staining (red), fluorescence in situ hybridization using Cy5-oligo(dT) probes (cyan) and Hoechst dye (blue). The arrows show transfected cells. Images were taken using the Leica Sp5 confocal microscope with a 63X objective lens and 2X zoom (B) Box plot of the fraction of nuclear to total Cy5-oligo(dT) fluorescence intensity in each cell. At least 50 cells were counted for each condition from two independent experiments. Data are mean ± SD. p > 0.05 (ns), p
    Figure Legend Snippet: CrPV-1A localizes to the nucleus and induces poly(A)+ RNA accumulation in the nucleus. (A) Confocal immunofluorescence images of S2 cells transfected with in vitro transcribed RNA encoding CrPV-1A, CrPV-1A(R146A), or CrPV-1A(F114A) for 16 hours. GFP fluorescence (green), CrPV-1A antibody staining (red), fluorescence in situ hybridization using Cy5-oligo(dT) probes (cyan) and Hoechst dye (blue). The arrows show transfected cells. Images were taken using the Leica Sp5 confocal microscope with a 63X objective lens and 2X zoom (B) Box plot of the fraction of nuclear to total Cy5-oligo(dT) fluorescence intensity in each cell. At least 50 cells were counted for each condition from two independent experiments. Data are mean ± SD. p > 0.05 (ns), p

    Techniques Used: Immunofluorescence, Transfection, In Vitro, Fluorescence, Staining, In Situ Hybridization, Microscopy

    RNA export modulates CrPV infection. (A) Fluorescence in situ hybridization using Cy5-oligo(dT) (blue) of S2 cells incubated with dsRNA targeting RNA export factor NXF1 or control GFP and Hoechst dye (blue), followed by mock infection or infection with wild-type or R146A mutant virus for 8 hours (MOI 10). (B) Viral yield from wild-type and mutant (R146A) CrPV infected S2 cells was accessed by fluorescence foci unit (FFU). Shown are averages from two independent experiments.
    Figure Legend Snippet: RNA export modulates CrPV infection. (A) Fluorescence in situ hybridization using Cy5-oligo(dT) (blue) of S2 cells incubated with dsRNA targeting RNA export factor NXF1 or control GFP and Hoechst dye (blue), followed by mock infection or infection with wild-type or R146A mutant virus for 8 hours (MOI 10). (B) Viral yield from wild-type and mutant (R146A) CrPV infected S2 cells was accessed by fluorescence foci unit (FFU). Shown are averages from two independent experiments.

    Techniques Used: Infection, Fluorescence, In Situ Hybridization, Incubation, Mutagenesis

    CrPV-1A expression inhibits stress granules in response to arsenite treatment. (A) Depiction of the CrPV genome with the structure of CrPV-1A protein (PDB 6C3R) ( below ) highlighting the domains selected for mutagenesis. ( B) Schematic of CrPV-1A-2A-GFP RNA containing the CrPV 5’ and 3’UTRs. (C) Confocal immunofluorescence images of S2 cells transfected with control 5’cap-GFP-poly (A)+, wild type or R146A mutant CrPV-1A-2A-GFP RNAs (16 hours) followed by one-hour treatment in the presence or absence of 500 µM sodium arsenite. The arrows show transfected cells. Shown are representative transfected cells detecting GFP fluorescence (green), Rin antibody staining (red), Hoechst dye staining for nucleus (blue) and merged images. Images were taken using the Leica Sp5 confocal microscope with a 63X objective lens and 2X zoom (D) Box plot showing the number Rin foci per cell. At least 50 cells were counted for each condition from three independent experiments. Data are mean ± SD. P > 0.05 (ns) p
    Figure Legend Snippet: CrPV-1A expression inhibits stress granules in response to arsenite treatment. (A) Depiction of the CrPV genome with the structure of CrPV-1A protein (PDB 6C3R) ( below ) highlighting the domains selected for mutagenesis. ( B) Schematic of CrPV-1A-2A-GFP RNA containing the CrPV 5’ and 3’UTRs. (C) Confocal immunofluorescence images of S2 cells transfected with control 5’cap-GFP-poly (A)+, wild type or R146A mutant CrPV-1A-2A-GFP RNAs (16 hours) followed by one-hour treatment in the presence or absence of 500 µM sodium arsenite. The arrows show transfected cells. Shown are representative transfected cells detecting GFP fluorescence (green), Rin antibody staining (red), Hoechst dye staining for nucleus (blue) and merged images. Images were taken using the Leica Sp5 confocal microscope with a 63X objective lens and 2X zoom (D) Box plot showing the number Rin foci per cell. At least 50 cells were counted for each condition from three independent experiments. Data are mean ± SD. P > 0.05 (ns) p

    Techniques Used: Expressing, Mutagenesis, Immunofluorescence, Transfection, Fluorescence, Staining, Microscopy

    17) Product Images from "Allele-specific expression changes dynamically during T cell activation in HLA and other autoimmune loci"

    Article Title: Allele-specific expression changes dynamically during T cell activation in HLA and other autoimmune loci

    Journal: Nature genetics

    doi: 10.1038/s41588-020-0579-4

    Scheme depicting HLA allelic expression quantification with HLA-personalized genome. In order to quantify robustly allele-specific expression in the highly polymorphic HLA genes, we first create an HLA-personalized genome per individual. We do this by inserting into the reference genome the cDNA sequences of each HLA allele as separate sequences (12 in total giv en that we sequenced or typed 6 HLA genes), and masking the exonic sequences corresponding to those cDNAs in chromosome 6 of the reference genome. Next, we map the RNA-seq reads to this HLA-personalized genome, we remove PCR duplicates and we count the number of uniquely mapped reads to each HLA cDNA allele.
    Figure Legend Snippet: Scheme depicting HLA allelic expression quantification with HLA-personalized genome. In order to quantify robustly allele-specific expression in the highly polymorphic HLA genes, we first create an HLA-personalized genome per individual. We do this by inserting into the reference genome the cDNA sequences of each HLA allele as separate sequences (12 in total giv en that we sequenced or typed 6 HLA genes), and masking the exonic sequences corresponding to those cDNAs in chromosome 6 of the reference genome. Next, we map the RNA-seq reads to this HLA-personalized genome, we remove PCR duplicates and we count the number of uniquely mapped reads to each HLA cDNA allele.

    Techniques Used: Expressing, RNA Sequencing Assay, Polymerase Chain Reaction

    18) Product Images from "Merkel Cell Polyomavirus Small Tumor Antigen Activates Matrix Metallopeptidase-9 Gene Expression for Cell Migration and Invasion"

    Article Title: Merkel Cell Polyomavirus Small Tumor Antigen Activates Matrix Metallopeptidase-9 Gene Expression for Cell Migration and Invasion

    Journal: bioRxiv

    doi: 10.1101/2020.03.02.974303

    MCV sT activates Matrix metalloproteinase 9 (MMP-9). (A) MCV sT expression results in upregulation of MMP-9 mRNA levels. Various cell lines (293, COS-7, MCC13 and U2OS) were transfected with either empty vector or MCV sT WT expressing plasmids to measure MMP-9 mRNA levels. After 48 h, total RNA was isolated and analyzed by RT-qPCR. (B) MCV sT upregulates MMP-9 transcription through the LSD. U2OS and MCC13 cells transfected with empty vector control, MCV sT WT and MCV sT LSDm expressing plasmids. Transcript levels of MMP-9 were analyzed using the comparative ΔΔCt method. (n = 3). Differences between means ( p value) were analyzed using a t-test with GraphPad Prism software. (C) MCV sT upregulates MMP-9 protein expression through the LSD. U2OS cells were transfected with empty vector, sT WT and sT LSDm expression plasmids. After 48 h, immunoblot analysis was performed to analyze expression of MMP-9, sT and α-tubulin (Ci). Densitometry quantification of immunoblots was carried out using the Image studio software and is shown as a fold change relative to the loading control α-tubulin (Cii). Data analyzed using three biological replicates per experiment (n = 3). (D) MCV sT reproducibly activates MMP-9 expression in MCC13.
    Figure Legend Snippet: MCV sT activates Matrix metalloproteinase 9 (MMP-9). (A) MCV sT expression results in upregulation of MMP-9 mRNA levels. Various cell lines (293, COS-7, MCC13 and U2OS) were transfected with either empty vector or MCV sT WT expressing plasmids to measure MMP-9 mRNA levels. After 48 h, total RNA was isolated and analyzed by RT-qPCR. (B) MCV sT upregulates MMP-9 transcription through the LSD. U2OS and MCC13 cells transfected with empty vector control, MCV sT WT and MCV sT LSDm expressing plasmids. Transcript levels of MMP-9 were analyzed using the comparative ΔΔCt method. (n = 3). Differences between means ( p value) were analyzed using a t-test with GraphPad Prism software. (C) MCV sT upregulates MMP-9 protein expression through the LSD. U2OS cells were transfected with empty vector, sT WT and sT LSDm expression plasmids. After 48 h, immunoblot analysis was performed to analyze expression of MMP-9, sT and α-tubulin (Ci). Densitometry quantification of immunoblots was carried out using the Image studio software and is shown as a fold change relative to the loading control α-tubulin (Cii). Data analyzed using three biological replicates per experiment (n = 3). (D) MCV sT reproducibly activates MMP-9 expression in MCC13.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Isolation, Quantitative RT-PCR, Software, Western Blot

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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 ∆QueC. Control (WT) and siEhDUF2419 trophozoites were cultivated in the presence of E. coli K12 or E. coli ∆QueC for 7 days (ration of 1 trophozoite:1000 bacteria). (1) WT trophozoites (2) queuine-treated WT trophozoites (3) WT trophozoites that were cultivated with E. coli K12 (4) WT trophozoites that were cultivated with E. coli ∆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 ∆QueC (9) E. coli K12 <t>RNA.</t> Data are from two biological replicates, each with two technical replicates. ** indicates p value
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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 ∆QueC. Control (WT) and siEhDUF2419 trophozoites were cultivated in the presence of E. coli K12 or E. coli ∆QueC for 7 days (ration of 1 trophozoite:1000 bacteria). (1) WT trophozoites (2) queuine-treated WT trophozoites (3) WT trophozoites that were cultivated with E. coli K12 (4) WT trophozoites that were cultivated with E. coli ∆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 ∆QueC (9) E. coli K12 RNA. Data are from two biological replicates, each with two technical replicates. ** indicates p value

    Journal: Cells

    Article Title: Queuine Salvaging in the Human Parasite Entamoeba histolytica

    doi: 10.3390/cells11162509

    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 ∆QueC. Control (WT) and siEhDUF2419 trophozoites were cultivated in the presence of E. coli K12 or E. coli ∆QueC for 7 days (ration of 1 trophozoite:1000 bacteria). (1) WT trophozoites (2) queuine-treated WT trophozoites (3) WT trophozoites that were cultivated with E. coli K12 (4) WT trophozoites that were cultivated with E. coli ∆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 ∆QueC (9) E. coli K12 RNA. Data are from two biological replicates, each with two technical replicates. ** indicates p value

    Article Snippet: Total RNA was extracted from E. histolytica trophozoites that were incubated with E. coli K12/∆QueC (a kind gift of Prof. Valérie de Crécy-Lagard, University of Florida, Gainesville, FL, USA) using the Monarch Total RNA Miniprep Kit (NEW ENGLAND BioLabs, Ornat, Nes Ziona, Israel).

    Techniques: Northern Blot

    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