rna clean  (Zymo Research)


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    Name:
    RNA Clean Concentrator 25
    Description:
    The RNA Clean Concentrator kits provide a simple and reliable method for the rapid preparation of high quality RT PCR ready DNA free R1013 R1014 RNA This simple procedure is based on the use of a unique single buffer system and Zymo Spin column technology that allows for selective recovery of total RNA 17 nt large RNAs 200 nt and or small RNAs 17 200 nt The procedure is easy Add binding buffer and ethanol to your sample then bind wash and elute ultra pure RNA The RNA can be eluted from the Zymo Spin IC Column in as little as 6 µl of RNase free water The highly concentrated purified RNA is suitable for all subsequent analyses and molecular manipulations
    Catalog Number:
    r1017
    Price:
    None
    Applications:
    RNA Purification
    Size:
    50 units
    Category:
    Life Science Reagents and Media
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    Structured Review

    Zymo Research rna clean
    Functional impact of macrophages on breast tumor cells. (A) Enriched biological processes as determined by GO term analysis of the <t>RNA</t> seq data from MΦ-infiltrated and non-infiltrated tumor spheroids. (B) 1 x 10 4 <t>MCF7</t> cells were seeded in a 96-well plate and incubated with supernatants from MCF7 cells or MΦs. Proliferation was assessed using an IncuCyte S3 system and is presented as relative increase in confluency. Data are presented as means ± SEM (n > 3, * p
    The RNA Clean Concentrator kits provide a simple and reliable method for the rapid preparation of high quality RT PCR ready DNA free R1013 R1014 RNA This simple procedure is based on the use of a unique single buffer system and Zymo Spin column technology that allows for selective recovery of total RNA 17 nt large RNAs 200 nt and or small RNAs 17 200 nt The procedure is easy Add binding buffer and ethanol to your sample then bind wash and elute ultra pure RNA The RNA can be eluted from the Zymo Spin IC Column in as little as 6 µl of RNase free water The highly concentrated purified RNA is suitable for all subsequent analyses and molecular manipulations
    https://www.bioz.com/result/rna clean/product/Zymo Research
    Average 99 stars, based on 199 article reviews
    Price from $9.99 to $1999.99
    rna clean - by Bioz Stars, 2020-08
    99/100 stars

    Images

    1) Product Images from "Macrophages attenuate the transcription of CYP1A1 in breast tumor cells and enhance their proliferation"

    Article Title: Macrophages attenuate the transcription of CYP1A1 in breast tumor cells and enhance their proliferation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0209694

    Functional impact of macrophages on breast tumor cells. (A) Enriched biological processes as determined by GO term analysis of the RNA seq data from MΦ-infiltrated and non-infiltrated tumor spheroids. (B) 1 x 10 4 MCF7 cells were seeded in a 96-well plate and incubated with supernatants from MCF7 cells or MΦs. Proliferation was assessed using an IncuCyte S3 system and is presented as relative increase in confluency. Data are presented as means ± SEM (n > 3, * p
    Figure Legend Snippet: Functional impact of macrophages on breast tumor cells. (A) Enriched biological processes as determined by GO term analysis of the RNA seq data from MΦ-infiltrated and non-infiltrated tumor spheroids. (B) 1 x 10 4 MCF7 cells were seeded in a 96-well plate and incubated with supernatants from MCF7 cells or MΦs. Proliferation was assessed using an IncuCyte S3 system and is presented as relative increase in confluency. Data are presented as means ± SEM (n > 3, * p

    Techniques Used: Functional Assay, RNA Sequencing Assay, Incubation

    Tumor cell-specific gene expression changes after macrophage infiltration. (A) Schematic overview of the experimental setup of tumor cell isolation for RNA seq. (B) Purity of tumor cells after removal of CD14 + cells from dissociated tumor spheroids was determined by FACS analysis of tumor cells (EpCAM + ) and immune cells (CD45 + ). Graph is representative of 3 independent experiments. The proportion of immune cells (CD45 + ) was quantified relative to all cells and is given as mean ± SEM (n = 3). (C) Top differentially expressed genes identified by RNA seq analysis of tumor cells from infiltrated relative to non-infiltrated MCF7 tumor spheroids.
    Figure Legend Snippet: Tumor cell-specific gene expression changes after macrophage infiltration. (A) Schematic overview of the experimental setup of tumor cell isolation for RNA seq. (B) Purity of tumor cells after removal of CD14 + cells from dissociated tumor spheroids was determined by FACS analysis of tumor cells (EpCAM + ) and immune cells (CD45 + ). Graph is representative of 3 independent experiments. The proportion of immune cells (CD45 + ) was quantified relative to all cells and is given as mean ± SEM (n = 3). (C) Top differentially expressed genes identified by RNA seq analysis of tumor cells from infiltrated relative to non-infiltrated MCF7 tumor spheroids.

    Techniques Used: Expressing, Cell Isolation, RNA Sequencing Assay, FACS

    2) Product Images from "Rapid and scalable profiling of nascent RNA with fastGRO"

    Article Title: Rapid and scalable profiling of nascent RNA with fastGRO

    Journal: bioRxiv

    doi: 10.1101/2020.01.24.916015

    Overview of fastGRO. In day 1, nuclei are isolated and in vitro run-on is performed in a solution containing 4-thio-UTP that is incorporated in nascent RNA. After isolation using Trizol and ethanol precipitation, RNA is fragmented and snap-frozen. In day 2, 4-thio-UTP containing RNA is biotinylated using either HPDP- (standard protocol) or MTS-biotin (fastGRO-LI protocol for low input sample) and recovered by immunoprecipitation using streptavidin-conjugated beads. Labeled RNA is recovered by elution in DTT solution, purified and used for NGS libraries preparation with commercially available kits.
    Figure Legend Snippet: Overview of fastGRO. In day 1, nuclei are isolated and in vitro run-on is performed in a solution containing 4-thio-UTP that is incorporated in nascent RNA. After isolation using Trizol and ethanol precipitation, RNA is fragmented and snap-frozen. In day 2, 4-thio-UTP containing RNA is biotinylated using either HPDP- (standard protocol) or MTS-biotin (fastGRO-LI protocol for low input sample) and recovered by immunoprecipitation using streptavidin-conjugated beads. Labeled RNA is recovered by elution in DTT solution, purified and used for NGS libraries preparation with commercially available kits.

    Techniques Used: Isolation, In Vitro, Ethanol Precipitation, Immunoprecipitation, Labeling, Purification, Next-Generation Sequencing

    3) Product Images from "Macrophages attenuate the transcription of CYP1A1 in breast tumor cells and enhance their proliferation"

    Article Title: Macrophages attenuate the transcription of CYP1A1 in breast tumor cells and enhance their proliferation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0209694

    Functional impact of macrophages on breast tumor cells. (A) Enriched biological processes as determined by GO term analysis of the RNA seq data from MΦ-infiltrated and non-infiltrated tumor spheroids. (B) 1 x 10 4 MCF7 cells were seeded in a 96-well plate and incubated with supernatants from MCF7 cells or MΦs. Proliferation was assessed using an IncuCyte S3 system and is presented as relative increase in confluency. Data are presented as means ± SEM (n > 3, * p
    Figure Legend Snippet: Functional impact of macrophages on breast tumor cells. (A) Enriched biological processes as determined by GO term analysis of the RNA seq data from MΦ-infiltrated and non-infiltrated tumor spheroids. (B) 1 x 10 4 MCF7 cells were seeded in a 96-well plate and incubated with supernatants from MCF7 cells or MΦs. Proliferation was assessed using an IncuCyte S3 system and is presented as relative increase in confluency. Data are presented as means ± SEM (n > 3, * p

    Techniques Used: Functional Assay, RNA Sequencing Assay, Incubation

    Tumor cell-specific gene expression changes after macrophage infiltration. (A) Schematic overview of the experimental setup of tumor cell isolation for RNA seq. (B) Purity of tumor cells after removal of CD14 + cells from dissociated tumor spheroids was determined by FACS analysis of tumor cells (EpCAM + ) and immune cells (CD45 + ). Graph is representative of 3 independent experiments. The proportion of immune cells (CD45 + ) was quantified relative to all cells and is given as mean ± SEM (n = 3). (C) Top differentially expressed genes identified by RNA seq analysis of tumor cells from infiltrated relative to non-infiltrated MCF7 tumor spheroids.
    Figure Legend Snippet: Tumor cell-specific gene expression changes after macrophage infiltration. (A) Schematic overview of the experimental setup of tumor cell isolation for RNA seq. (B) Purity of tumor cells after removal of CD14 + cells from dissociated tumor spheroids was determined by FACS analysis of tumor cells (EpCAM + ) and immune cells (CD45 + ). Graph is representative of 3 independent experiments. The proportion of immune cells (CD45 + ) was quantified relative to all cells and is given as mean ± SEM (n = 3). (C) Top differentially expressed genes identified by RNA seq analysis of tumor cells from infiltrated relative to non-infiltrated MCF7 tumor spheroids.

    Techniques Used: Expressing, Cell Isolation, RNA Sequencing Assay, FACS

    4) Product Images from "Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair"

    Article Title: Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair

    Journal: eLife

    doi: 10.7554/eLife.33761

    Covalent linkage of the DNA repair template to the Cas9 RNP complex. ( a ) Quantification of HDR rates with DNA repair templates of different lengths by FACS. ( b ) Quantification of sgRNA Sp Cas9 (mutRFP) editing efficiency in the reporter cell line by FACS. ( c ) SYBR-Gold stained denaturing PAGE gel. Proportion of shifted 81-mer amino-modified oligos after the coupling reaction with different concentrations of BG-GLA-NHS building blocks. ( d ) Negative ion ESI mass spectra of HPLC purified BG-coupled (top panel) and uncoupled (bottom panel) oligos shown in Figure 3b . ( e,f ) SDS-PAGE gels of Sp Cas9-SNAP ( e ) and the Sa dCas9-SNAP ( f ) proteins labeled with BG-Vista Green (upper panels). Subsequently gels were silver stained for protein detection (lower panels). ( g,h ) Denaturing RNA gels (2% MOPS) with in vitro transcribed sgRNAs. ( g ) sgRNA used to create the DSB in the mutRFP locus (sgRNA Sp Cas9 (mutRFP)). ( h ) sgRNAs used to bind catalytically inactive Sa dCas9 to regions upstream (sgRNA Sa dCas9 (mutRFP)−1 and sgRNA Sa dCas9 (mutRFP)−2) and downstream (sgRNA Sa dCas9 (mutRFP)−3 and sgRNA Sa dCas9 (mutRFP)−4) of the mutRFP locus. Box and whiskers plot: min to max (**p
    Figure Legend Snippet: Covalent linkage of the DNA repair template to the Cas9 RNP complex. ( a ) Quantification of HDR rates with DNA repair templates of different lengths by FACS. ( b ) Quantification of sgRNA Sp Cas9 (mutRFP) editing efficiency in the reporter cell line by FACS. ( c ) SYBR-Gold stained denaturing PAGE gel. Proportion of shifted 81-mer amino-modified oligos after the coupling reaction with different concentrations of BG-GLA-NHS building blocks. ( d ) Negative ion ESI mass spectra of HPLC purified BG-coupled (top panel) and uncoupled (bottom panel) oligos shown in Figure 3b . ( e,f ) SDS-PAGE gels of Sp Cas9-SNAP ( e ) and the Sa dCas9-SNAP ( f ) proteins labeled with BG-Vista Green (upper panels). Subsequently gels were silver stained for protein detection (lower panels). ( g,h ) Denaturing RNA gels (2% MOPS) with in vitro transcribed sgRNAs. ( g ) sgRNA used to create the DSB in the mutRFP locus (sgRNA Sp Cas9 (mutRFP)). ( h ) sgRNAs used to bind catalytically inactive Sa dCas9 to regions upstream (sgRNA Sa dCas9 (mutRFP)−1 and sgRNA Sa dCas9 (mutRFP)−2) and downstream (sgRNA Sa dCas9 (mutRFP)−3 and sgRNA Sa dCas9 (mutRFP)−4) of the mutRFP locus. Box and whiskers plot: min to max (**p

    Techniques Used: FACS, Staining, Polyacrylamide Gel Electrophoresis, Modification, High Performance Liquid Chromatography, Purification, SDS Page, Labeling, In Vitro

    5) Product Images from "CiBER-seq dissects genetic networks by quantitative CRISPRi profiling of expression phenotypes"

    Article Title: CiBER-seq dissects genetic networks by quantitative CRISPRi profiling of expression phenotypes

    Journal: bioRxiv

    doi: 10.1101/2020.03.29.015057

    Barcoded expression reporters accurately link guide RNAs with transcriptional phenotypes. ( A ) Schematic of synthetic ZEM transcription factor and P(Z) promoter. ( B , C ) Expression measurements by (B) fluorescence and (C) RNA/DNA barcode count ratio show quantitative agreement. Inducer conditions for barcode sequencing in (C) are color-coded and marked with dots on the graph in (B). ( D ) Schematic of guide RNA effects on the synthetic transcription factor / promoter pair. ( E ) Fluorescent output is negatively correlated with guide RNA induction level. ( F ) Barcode sequencing expression measurements reflect effects of knockdown. ( G ) Schematic of CiBER-Seq profiling experiment.
    Figure Legend Snippet: Barcoded expression reporters accurately link guide RNAs with transcriptional phenotypes. ( A ) Schematic of synthetic ZEM transcription factor and P(Z) promoter. ( B , C ) Expression measurements by (B) fluorescence and (C) RNA/DNA barcode count ratio show quantitative agreement. Inducer conditions for barcode sequencing in (C) are color-coded and marked with dots on the graph in (B). ( D ) Schematic of guide RNA effects on the synthetic transcription factor / promoter pair. ( E ) Fluorescent output is negatively correlated with guide RNA induction level. ( F ) Barcode sequencing expression measurements reflect effects of knockdown. ( G ) Schematic of CiBER-Seq profiling experiment.

    Techniques Used: Expressing, Fluorescence, Sequencing

    Related Articles

    Clone Assay:

    Article Title: Kif18a regulates Sirt2-mediated tubulin acetylation for spindle organization during mouse oocyte meiosis
    Article Snippet: .. Vector (pcDNA3.1) cloning with Flag-tubulin substitution mutants (K40R) was conducted by the StarMut site-directed mutagenesis kit (GenStar, Cat#T111-01). mRNA was synthesized from linearized plasmid using the HiScribe T7 high yield RNA synthesis kit (NEB), then capped with m7G(5′)ppp(5′)G (NEB), tailed with a poly(A) polymerase tailing kit (Epicentre), and purified with the RNA clean & concentrator-25 kit (Zymo Research). .. Kif18a siRNA injection Kif18a siRNA microinjection was used to knock down Kif18a in mouse oocytes.

    Synthesized:

    Article Title: Kif18a regulates Sirt2-mediated tubulin acetylation for spindle organization during mouse oocyte meiosis
    Article Snippet: .. Vector (pcDNA3.1) cloning with Flag-tubulin substitution mutants (K40R) was conducted by the StarMut site-directed mutagenesis kit (GenStar, Cat#T111-01). mRNA was synthesized from linearized plasmid using the HiScribe T7 high yield RNA synthesis kit (NEB), then capped with m7G(5′)ppp(5′)G (NEB), tailed with a poly(A) polymerase tailing kit (Epicentre), and purified with the RNA clean & concentrator-25 kit (Zymo Research). .. Kif18a siRNA injection Kif18a siRNA microinjection was used to knock down Kif18a in mouse oocytes.

    Mutagenesis:

    Article Title: Kif18a regulates Sirt2-mediated tubulin acetylation for spindle organization during mouse oocyte meiosis
    Article Snippet: .. Vector (pcDNA3.1) cloning with Flag-tubulin substitution mutants (K40R) was conducted by the StarMut site-directed mutagenesis kit (GenStar, Cat#T111-01). mRNA was synthesized from linearized plasmid using the HiScribe T7 high yield RNA synthesis kit (NEB), then capped with m7G(5′)ppp(5′)G (NEB), tailed with a poly(A) polymerase tailing kit (Epicentre), and purified with the RNA clean & concentrator-25 kit (Zymo Research). .. Kif18a siRNA injection Kif18a siRNA microinjection was used to knock down Kif18a in mouse oocytes.

    Isolation:

    Article Title: Macrophages attenuate the transcription of CYP1A1 in breast tumor cells and enhance their proliferation
    Article Snippet: .. Briefly, RNA was isolated out of 100 μl MCF7 tumor cell lysates using the RNA Clean and Concentrator-25 kit (Zymo Research). rRNA was removed using the RiboZero Gold rRNA Removal kit (Human/Mouse/Rat, Illumina). .. After heat fragmentation, end repair, and ligation of 3’ adapters, the RNA was reverse transcribed.

    Labeling:

    Article Title: Dissection of specific binding of HIV-1 Gag to the 'packaging signal' in viral RNA
    Article Snippet: .. To purify the RNAs the samples were run through three consecutive illustra microspin G-50 columns (GE Healthcare Life Sciences, Pittsburgh, PA USA) per 35 μL of labeling reaction, concentrated with an RNA clean and concentrator-25 kit (Zymo Research, Irvine, CA USA) and eluted with double-autoclaved double deionized water (dd-Water). .. The labeling yield was determined according to the manufacturer’s instructions with a UV-Vis NanoDrop 1000 and the integrity of the RNAs was determined by gel electrophoresis (6% polyacrylamide/TBE-UREA).

    Purification:

    Article Title: Kif18a regulates Sirt2-mediated tubulin acetylation for spindle organization during mouse oocyte meiosis
    Article Snippet: .. Vector (pcDNA3.1) cloning with Flag-tubulin substitution mutants (K40R) was conducted by the StarMut site-directed mutagenesis kit (GenStar, Cat#T111-01). mRNA was synthesized from linearized plasmid using the HiScribe T7 high yield RNA synthesis kit (NEB), then capped with m7G(5′)ppp(5′)G (NEB), tailed with a poly(A) polymerase tailing kit (Epicentre), and purified with the RNA clean & concentrator-25 kit (Zymo Research). .. Kif18a siRNA injection Kif18a siRNA microinjection was used to knock down Kif18a in mouse oocytes.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Kif18a regulates Sirt2-mediated tubulin acetylation for spindle organization during mouse oocyte meiosis
    Article Snippet: .. Vector (pcDNA3.1) cloning with Flag-tubulin substitution mutants (K40R) was conducted by the StarMut site-directed mutagenesis kit (GenStar, CatT111-01). mRNA was synthesized from linearized plasmid using the HiScribe T7 high yield RNA synthesis kit (NEB), then capped with m7G(5′)ppp(5′)G (NEB), tailed with a poly(A) polymerase tailing kit (Epicentre), and purified with the RNA clean & concentrator-25 kit (Zymo Research). .. Kif18a siRNA injection Kif18a siRNA microinjection was used to knock down Kif18a in mouse oocytes.

    Plasmid Preparation:

    Article Title: Kif18a regulates Sirt2-mediated tubulin acetylation for spindle organization during mouse oocyte meiosis
    Article Snippet: .. Vector (pcDNA3.1) cloning with Flag-tubulin substitution mutants (K40R) was conducted by the StarMut site-directed mutagenesis kit (GenStar, Cat#T111-01). mRNA was synthesized from linearized plasmid using the HiScribe T7 high yield RNA synthesis kit (NEB), then capped with m7G(5′)ppp(5′)G (NEB), tailed with a poly(A) polymerase tailing kit (Epicentre), and purified with the RNA clean & concentrator-25 kit (Zymo Research). .. Kif18a siRNA injection Kif18a siRNA microinjection was used to knock down Kif18a in mouse oocytes.

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    Zymo Research rna clean
    Functional impact of macrophages on breast tumor cells. (A) Enriched biological processes as determined by GO term analysis of the <t>RNA</t> seq data from MΦ-infiltrated and non-infiltrated tumor spheroids. (B) 1 x 10 4 <t>MCF7</t> cells were seeded in a 96-well plate and incubated with supernatants from MCF7 cells or MΦs. Proliferation was assessed using an IncuCyte S3 system and is presented as relative increase in confluency. Data are presented as means ± SEM (n > 3, * p
    Rna Clean, supplied by Zymo Research, used in various techniques. Bioz Stars score: 99/100, based on 199 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna clean/product/Zymo Research
    Average 99 stars, based on 199 article reviews
    Price from $9.99 to $1999.99
    rna clean - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    99
    Zymo Research rna clean concentrator
    Measurement of R-loop formation by LbCas12a using a magnetic tweezers assay. ( A ) DNA protospacer sequence (black) and CRISPR <t>RNA</t> <t>(crRNA)</t> showing the G-residues from in vitro transcription (brown), the pseudoknot (blue) and spacer (red). ( B ) Principle of the MT assay. See main text. ( C ) R-loop cycling experiment (1 turn s −1 ) in the presence of 5 nM Cas12a:crRNA. Raw DNA length taken at 60 Hz (grey). Data smoothed by a 1 Hz moving average (dark colors). DNA is negatively supercoiled at 0.3 pN (red) to induce R-loop formation (in), followed by positive supercoiling to probe R-loop formation (blue), resulting in R-loop dissociation (out). Rot 0 are points where DNA turns are zero. ( D ) Overlay of R-loop cycles ( N = 22) for negative supercoiling (in events) and positive supercoiling (out events). Cycles without Cas12a are in grey. Data was smoothed by a 1 Hz moving average. ( i ) and ( ii ) show rotation curve shifts due to captured R-loops. ( E ) Rotation curve shift due to R-loop events ( i ). Average = 1.87 ± 0.27 turns (errors = SD). ( F ) Examples of repetitive R-loop formation cycling (at 10 turns s −1 ) to measure R-loop formation times. Raw and 1 Hz smoothed data are shown. ( G ) Mean R-loop formation/dissociation times and standard error ( N = 40 to 52) as a function of torque [ 29 ]. Solid lines are fits to Equation (1) ( Table 1 ) [ 27 ]. ( H ) Inverted cumulative probability over time for R-loop formation (left) and dissociation (right) used to calculate mean times in panel F.
    Rna Clean Concentrator, supplied by Zymo Research, used in various techniques. Bioz Stars score: 99/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna clean concentrator/product/Zymo Research
    Average 99 stars, based on 31 article reviews
    Price from $9.99 to $1999.99
    rna clean concentrator - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

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    Functional impact of macrophages on breast tumor cells. (A) Enriched biological processes as determined by GO term analysis of the RNA seq data from MΦ-infiltrated and non-infiltrated tumor spheroids. (B) 1 x 10 4 MCF7 cells were seeded in a 96-well plate and incubated with supernatants from MCF7 cells or MΦs. Proliferation was assessed using an IncuCyte S3 system and is presented as relative increase in confluency. Data are presented as means ± SEM (n > 3, * p

    Journal: PLoS ONE

    Article Title: Macrophages attenuate the transcription of CYP1A1 in breast tumor cells and enhance their proliferation

    doi: 10.1371/journal.pone.0209694

    Figure Lengend Snippet: Functional impact of macrophages on breast tumor cells. (A) Enriched biological processes as determined by GO term analysis of the RNA seq data from MΦ-infiltrated and non-infiltrated tumor spheroids. (B) 1 x 10 4 MCF7 cells were seeded in a 96-well plate and incubated with supernatants from MCF7 cells or MΦs. Proliferation was assessed using an IncuCyte S3 system and is presented as relative increase in confluency. Data are presented as means ± SEM (n > 3, * p

    Article Snippet: Briefly, RNA was isolated out of 100 μl MCF7 tumor cell lysates using the RNA Clean and Concentrator-25 kit (Zymo Research). rRNA was removed using the RiboZero Gold rRNA Removal kit (Human/Mouse/Rat, Illumina).

    Techniques: Functional Assay, RNA Sequencing Assay, Incubation

    Tumor cell-specific gene expression changes after macrophage infiltration. (A) Schematic overview of the experimental setup of tumor cell isolation for RNA seq. (B) Purity of tumor cells after removal of CD14 + cells from dissociated tumor spheroids was determined by FACS analysis of tumor cells (EpCAM + ) and immune cells (CD45 + ). Graph is representative of 3 independent experiments. The proportion of immune cells (CD45 + ) was quantified relative to all cells and is given as mean ± SEM (n = 3). (C) Top differentially expressed genes identified by RNA seq analysis of tumor cells from infiltrated relative to non-infiltrated MCF7 tumor spheroids.

    Journal: PLoS ONE

    Article Title: Macrophages attenuate the transcription of CYP1A1 in breast tumor cells and enhance their proliferation

    doi: 10.1371/journal.pone.0209694

    Figure Lengend Snippet: Tumor cell-specific gene expression changes after macrophage infiltration. (A) Schematic overview of the experimental setup of tumor cell isolation for RNA seq. (B) Purity of tumor cells after removal of CD14 + cells from dissociated tumor spheroids was determined by FACS analysis of tumor cells (EpCAM + ) and immune cells (CD45 + ). Graph is representative of 3 independent experiments. The proportion of immune cells (CD45 + ) was quantified relative to all cells and is given as mean ± SEM (n = 3). (C) Top differentially expressed genes identified by RNA seq analysis of tumor cells from infiltrated relative to non-infiltrated MCF7 tumor spheroids.

    Article Snippet: Briefly, RNA was isolated out of 100 μl MCF7 tumor cell lysates using the RNA Clean and Concentrator-25 kit (Zymo Research). rRNA was removed using the RiboZero Gold rRNA Removal kit (Human/Mouse/Rat, Illumina).

    Techniques: Expressing, Cell Isolation, RNA Sequencing Assay, FACS

    Measurement of R-loop formation by LbCas12a using a magnetic tweezers assay. ( A ) DNA protospacer sequence (black) and CRISPR RNA (crRNA) showing the G-residues from in vitro transcription (brown), the pseudoknot (blue) and spacer (red). ( B ) Principle of the MT assay. See main text. ( C ) R-loop cycling experiment (1 turn s −1 ) in the presence of 5 nM Cas12a:crRNA. Raw DNA length taken at 60 Hz (grey). Data smoothed by a 1 Hz moving average (dark colors). DNA is negatively supercoiled at 0.3 pN (red) to induce R-loop formation (in), followed by positive supercoiling to probe R-loop formation (blue), resulting in R-loop dissociation (out). Rot 0 are points where DNA turns are zero. ( D ) Overlay of R-loop cycles ( N = 22) for negative supercoiling (in events) and positive supercoiling (out events). Cycles without Cas12a are in grey. Data was smoothed by a 1 Hz moving average. ( i ) and ( ii ) show rotation curve shifts due to captured R-loops. ( E ) Rotation curve shift due to R-loop events ( i ). Average = 1.87 ± 0.27 turns (errors = SD). ( F ) Examples of repetitive R-loop formation cycling (at 10 turns s −1 ) to measure R-loop formation times. Raw and 1 Hz smoothed data are shown. ( G ) Mean R-loop formation/dissociation times and standard error ( N = 40 to 52) as a function of torque [ 29 ]. Solid lines are fits to Equation (1) ( Table 1 ) [ 27 ]. ( H ) Inverted cumulative probability over time for R-loop formation (left) and dissociation (right) used to calculate mean times in panel F.

    Journal: Genes

    Article Title: The Effect of DNA Topology on Observed Rates of R-Loop Formation and DNA Strand Cleavage by CRISPR Cas12a

    doi: 10.3390/genes10020169

    Figure Lengend Snippet: Measurement of R-loop formation by LbCas12a using a magnetic tweezers assay. ( A ) DNA protospacer sequence (black) and CRISPR RNA (crRNA) showing the G-residues from in vitro transcription (brown), the pseudoknot (blue) and spacer (red). ( B ) Principle of the MT assay. See main text. ( C ) R-loop cycling experiment (1 turn s −1 ) in the presence of 5 nM Cas12a:crRNA. Raw DNA length taken at 60 Hz (grey). Data smoothed by a 1 Hz moving average (dark colors). DNA is negatively supercoiled at 0.3 pN (red) to induce R-loop formation (in), followed by positive supercoiling to probe R-loop formation (blue), resulting in R-loop dissociation (out). Rot 0 are points where DNA turns are zero. ( D ) Overlay of R-loop cycles ( N = 22) for negative supercoiling (in events) and positive supercoiling (out events). Cycles without Cas12a are in grey. Data was smoothed by a 1 Hz moving average. ( i ) and ( ii ) show rotation curve shifts due to captured R-loops. ( E ) Rotation curve shift due to R-loop events ( i ). Average = 1.87 ± 0.27 turns (errors = SD). ( F ) Examples of repetitive R-loop formation cycling (at 10 turns s −1 ) to measure R-loop formation times. Raw and 1 Hz smoothed data are shown. ( G ) Mean R-loop formation/dissociation times and standard error ( N = 40 to 52) as a function of torque [ 29 ]. Solid lines are fits to Equation (1) ( Table 1 ) [ 27 ]. ( H ) Inverted cumulative probability over time for R-loop formation (left) and dissociation (right) used to calculate mean times in panel F.

    Article Snippet: The samples were subjected to the optional DNase I treatment, and the crRNA purified using an RNA Clean & Concentrator (Zymo Research, Irvine, CA, USA).

    Techniques: Sequencing, CRISPR, In Vitro

    Nm-seq, a method based on oxidative cleavage for mapping 2′- O -methylation with base precision. ( a ) Schematic illustration. Fragmented RNA (I) is subjected to iterative oxidation–elimination–dephosphorylation (OED) cycles that remove 2′-hydroxylated nucleotides in the 3′-to-5′ direction to expose internal Nm sites at 3′ ends of fragments (II). Fragments ending with 2′-hydroxyl are then blocked by an incomplete cycle (OE, III), followed by adaptor ligation and library construction (IV). Paired-end sequencing (V) produces an asymmetric coverage profile, the uniform 3′ end of which corresponds to an Nm site shown in red below (G). Red triangles, Nm sites; n and gray shadow, number of OED cycles equals number of removed nucleotides, except when Nm is encountered; x, 3′-monophosphates representing blocked ends; blue and orange lines, 5′ and 3′ adaptors, respectively. ( b ) Chemical structures of 2′- O -methylated and 2′-hydroxylated RNA polymers at intermediate steps (I, II, III) of the method. ( c ) MALDI-TOF spectra of model RNA oligonucleotides, 3′-modified (red) and unmodified (blue), produced at intermediate steps listed on the right. AU, arbitrary units. ( d ) OE effectively blocks 3′ adaptor ligation to 2′-hydroxylated but not to 2′- O -methylated 3′ ends. N, RNA model ending with 2′-hydroxyl; Nm, RNA model ending with 2′- O -methyl; Np, RNA model ending with 3′ monophosphate.

    Journal: Nature methods

    Article Title: Nm-seq maps 2′-O-methylation sites in human mRNA with base precision

    doi: 10.1038/nmeth.4294

    Figure Lengend Snippet: Nm-seq, a method based on oxidative cleavage for mapping 2′- O -methylation with base precision. ( a ) Schematic illustration. Fragmented RNA (I) is subjected to iterative oxidation–elimination–dephosphorylation (OED) cycles that remove 2′-hydroxylated nucleotides in the 3′-to-5′ direction to expose internal Nm sites at 3′ ends of fragments (II). Fragments ending with 2′-hydroxyl are then blocked by an incomplete cycle (OE, III), followed by adaptor ligation and library construction (IV). Paired-end sequencing (V) produces an asymmetric coverage profile, the uniform 3′ end of which corresponds to an Nm site shown in red below (G). Red triangles, Nm sites; n and gray shadow, number of OED cycles equals number of removed nucleotides, except when Nm is encountered; x, 3′-monophosphates representing blocked ends; blue and orange lines, 5′ and 3′ adaptors, respectively. ( b ) Chemical structures of 2′- O -methylated and 2′-hydroxylated RNA polymers at intermediate steps (I, II, III) of the method. ( c ) MALDI-TOF spectra of model RNA oligonucleotides, 3′-modified (red) and unmodified (blue), produced at intermediate steps listed on the right. AU, arbitrary units. ( d ) OE effectively blocks 3′ adaptor ligation to 2′-hydroxylated but not to 2′- O -methylated 3′ ends. N, RNA model ending with 2′-hydroxyl; Nm, RNA model ending with 2′- O -methyl; Np, RNA model ending with 3′ monophosphate.

    Article Snippet: The reaction was quenched by ethylene glycol, and the oligonucleotides were column purified (RNA Clean & Concentrator, Zymo) and analyzed by MALDI-TOF MS. Products were further dephosphorylated by Shrimp Alkaline Phosphatase (New England BioLabs, 40 units ml−1 ) at 37 °C for 30 min, column purified (RNA Clean & Concentrator, Zymo) and analyzed by MALDI-TOF MS.

    Techniques: Methylation, De-Phosphorylation Assay, Ligation, Sequencing, Modification, Produced

    Quality assessment metrics for RNA-Seq data. Box plots representing ( a ) GC content (%) and ( b ) Phred quality score distribution over all reads across all samples in each base (i.e., sequencing cycle). The box and horizontal bar represent the interquartile range and median of the ( a ) GC content and ( b ) median of Phred quality score over all reads. ( c ) Box plot representing the percentage of reads ( y -axis) that appear N times ( x -axis) relative to the number of unique reads from each sequencing sample across all samples. ( d ) The percentage of reads mapped (ratio, Mean ±s.e., n =32 in normal organs or 16 in sexual organs) to genomic regions, AceView exons, ERCCs and rRNA in each organ.

    Journal: Scientific Data

    Article Title: Comprehensive RNA-Seq transcriptomic profiling across 11 organs, 4 ages, and 2 sexes of Fischer 344 rats

    doi: 10.1038/sdata.2014.13

    Figure Lengend Snippet: Quality assessment metrics for RNA-Seq data. Box plots representing ( a ) GC content (%) and ( b ) Phred quality score distribution over all reads across all samples in each base (i.e., sequencing cycle). The box and horizontal bar represent the interquartile range and median of the ( a ) GC content and ( b ) median of Phred quality score over all reads. ( c ) Box plot representing the percentage of reads ( y -axis) that appear N times ( x -axis) relative to the number of unique reads from each sequencing sample across all samples. ( d ) The percentage of reads mapped (ratio, Mean ±s.e., n =32 in normal organs or 16 in sexual organs) to genomic regions, AceView exons, ERCCs and rRNA in each organ.

    Article Snippet: The rRNA-depleted RNA was purified using the RNA Clean & Concentrator Column (Zymo Research), which recovered all rRNA-depleted RNA, including small RNA.

    Techniques: RNA Sequencing Assay, Sequencing