monarch total rna miniprep kit  (New England Biolabs)

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

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

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

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

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

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

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

Journal: bioRxiv

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

doi: 10.1101/2022.05.19.492599

Figure Lengend 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

Article Snippet: cDNA synthesis and quantitative real time PCR Total RNA was extracted from cells using Monarch total RNA Miniprep kit (NEB). cDNA synthesis was performed using LunascriptTM RT Supermix Kit (NEB) as per manufacturer’s protocol. qRT PCR was performed using Luna Universal qPCR master mix (NEB) as per manufacturer’s protocol.

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

Journal: bioRxiv

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

doi: 10.1101/2022.05.19.492599

Figure Lengend 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.

Article Snippet: cDNA synthesis and quantitative real time PCR Total RNA was extracted from cells using Monarch total RNA Miniprep kit (NEB). cDNA synthesis was performed using LunascriptTM RT Supermix Kit (NEB) as per manufacturer’s protocol. qRT PCR was performed using Luna Universal qPCR master mix (NEB) as per manufacturer’s protocol.

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

Journal: bioRxiv

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

doi: 10.1101/2022.05.19.492599

Figure Lengend 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

Article Snippet: cDNA synthesis and quantitative real time PCR Total RNA was extracted from cells using Monarch total RNA Miniprep kit (NEB). cDNA synthesis was performed using LunascriptTM RT Supermix Kit (NEB) as per manufacturer’s protocol. qRT PCR was performed using Luna Universal qPCR master mix (NEB) as per manufacturer’s protocol.

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

Journal: bioRxiv

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

doi: 10.1101/2021.12.28.474323

Figure Lengend 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.

Article Snippet: RT-qPCR experiment Total RNA was extracted from cell-free expression reactions with a kit (NEB #T2010), following the manufacturer’s instructions.

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