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    Thermo Fisher dna high sensitivity qubit kit
    Extracellular <t>DNA</t> is enclosed mostly in L-EVs in plasma. ( a) 50 or 100 μg of PC3 EVs was spiked in 1 ml of normal plasma. Plasma EVs were isolated, EV DNA was extracted and (b) quantified using HS dsDNA <t>Qubit</t> Assay. (c) 50 or 100 μg of PC3 EVs was spiked in 1 ml of normal plasma. Plasma cell-free (cf)DNA was extracted and (d) quantified using HS dsDNA Qubit Assay. (e) Representative bioluminescent images showing progressive bone and visceral metastasis following intracardial injection of 1 × 10 7 luciferase-labelled PC3 cells in NOD/SCID mice (left). The bioluminescent signal was quantified weekly and was measured as radiance in p/sec/cm 2 /sr (right). (f) SCNV of MYC, AKT1, PTEN, PTK2 and KLF10 in plasma-derived L-EVs was assessed by dPCR, demonstrating that L-EVs in the plasma of a mouse model of bone metastases report tumour-specific SCNV. Copy number of each target gene was normalized to gene reference RNAse P. N = 3 for each data point.
    Dna High Sensitivity Qubit Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 82/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna high sensitivity qubit kit/product/Thermo Fisher
    Average 82 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    dna high sensitivity qubit kit - by Bioz Stars, 2019-10
    82/100 stars
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    99
    Agilent technologies high sensitivity dna analysis kit
    Size preference of NGS and titration of transposase. Profiles of an NGS library are compared: Panel A shows what was loaded onto MiSeq (analyzed by Bioanalyzer) and Panel B shows what was actually sequenced. Short fragments are more efficient in forming clusters. Panel C shows profiles of the libraries generated with various ratios of transposon to <t>DNA.</t> Traces 1–8 represent tagmentation reactions of 4.44 ng of human DNA by 0.023 μL, 0.047 μL, 0.093 μL, 0.19 μL, 0.38 μL, 0.75 μL, 1.5 μL, and 3 μL of Mu transposon respectively (details in Materials and methods ). Reactions represented by Traces 6, 7, and 8 were under saturating conditions.
    High Sensitivity Dna Analysis Kit, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 99/100, based on 584 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/high sensitivity dna analysis kit/product/Agilent technologies
    Average 99 stars, based on 584 article reviews
    Price from $9.99 to $1999.99
    high sensitivity dna analysis kit - by Bioz Stars, 2019-10
    99/100 stars
      Buy from Supplier

    Image Search Results


    Extracellular DNA is enclosed mostly in L-EVs in plasma. ( a) 50 or 100 μg of PC3 EVs was spiked in 1 ml of normal plasma. Plasma EVs were isolated, EV DNA was extracted and (b) quantified using HS dsDNA Qubit Assay. (c) 50 or 100 μg of PC3 EVs was spiked in 1 ml of normal plasma. Plasma cell-free (cf)DNA was extracted and (d) quantified using HS dsDNA Qubit Assay. (e) Representative bioluminescent images showing progressive bone and visceral metastasis following intracardial injection of 1 × 10 7 luciferase-labelled PC3 cells in NOD/SCID mice (left). The bioluminescent signal was quantified weekly and was measured as radiance in p/sec/cm 2 /sr (right). (f) SCNV of MYC, AKT1, PTEN, PTK2 and KLF10 in plasma-derived L-EVs was assessed by dPCR, demonstrating that L-EVs in the plasma of a mouse model of bone metastases report tumour-specific SCNV. Copy number of each target gene was normalized to gene reference RNAse P. N = 3 for each data point.

    Journal: Journal of Extracellular Vesicles

    Article Title: Large extracellular vesicles carry most of the tumour DNA circulating in prostate cancer patient plasma

    doi: 10.1080/20013078.2018.1505403

    Figure Lengend Snippet: Extracellular DNA is enclosed mostly in L-EVs in plasma. ( a) 50 or 100 μg of PC3 EVs was spiked in 1 ml of normal plasma. Plasma EVs were isolated, EV DNA was extracted and (b) quantified using HS dsDNA Qubit Assay. (c) 50 or 100 μg of PC3 EVs was spiked in 1 ml of normal plasma. Plasma cell-free (cf)DNA was extracted and (d) quantified using HS dsDNA Qubit Assay. (e) Representative bioluminescent images showing progressive bone and visceral metastasis following intracardial injection of 1 × 10 7 luciferase-labelled PC3 cells in NOD/SCID mice (left). The bioluminescent signal was quantified weekly and was measured as radiance in p/sec/cm 2 /sr (right). (f) SCNV of MYC, AKT1, PTEN, PTK2 and KLF10 in plasma-derived L-EVs was assessed by dPCR, demonstrating that L-EVs in the plasma of a mouse model of bone metastases report tumour-specific SCNV. Copy number of each target gene was normalized to gene reference RNAse P. N = 3 for each data point.

    Article Snippet: Final library quality control was performed using the DNA High Sensitivity Qubit Kit (Invitrogen), the Bioanalyzer High Sensitivity Chip Kit (Agilent) and the 7900HT Fast qPCR machine (Applied Biosystems). qPCR was performed using the Illumina Universal Library Quantification Kit from KAPA Biosystems.

    Techniques: Isolation, HS DSDNA Qubit Assay, Injection, Luciferase, Mouse Assay, Size-exclusion Chromatography, Derivative Assay, Digital PCR

    Most extracellular DNA is packaged into L-EVs . (a) Tunable resistive pulse sensing (TRPS, qNano) using two different pore membranes (NP4000 and NP200) identified as L-EVs (left) and S-EVs (right) derived from PC3 cells. NP4000 membrane, which can detect particles with a diameter between 1.0 and 6.0 μm, was used for quantitation of L-EVs, while NP200 membrane, which can detect particles with a diameter between 60 and 400 nm, was used for quantitation of S-EVs. (b) Protein lysates from L-EVs and S-EVs purified by iodixanol density gradient (at 1.10 and 1.15 g/ml) were blotted with LO markers HSPA5 and CK18, and with Exo marker CD81. (c) Total DNA was quantified by Qubit Fluorometer in L-EVs and S-EVs isolated from PC3 and U87 cell lines. The plot shows the DNA ratio between L-EVs and S-EVs. (d) Double stranded (ds)DNA was quantified by High Sensitivity (HS) dsDNA Qubit Assay in L-EVs and S-EVs isolated from 1 ml of plasma from patients with mCRPC ( n = 40) and cancer-free individuals ( n = 6). (e) Quantification of both protein and DNA content in L-EVs and S-EVs isolated from conditioned media of 12.6 × 10 7 PC3 cells. (f) Single stranded (ss) and dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Exonuclease III, were quantified by Qubit. (g) Chip-based capillary electrophoresis (Bioanalyzer) showing the presence of dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Endonuclease III. L-EVs contain abundant DNA with a large peak around 10 kbp. Conversely, the amount of DNA in S-EVs is negligible. (h) ss- and dsDNA in PC3-derived L-EVs and S-EVs were quantified by Qubit after treatment with nucleases (DNase I and Exonuclease III) with or without addition of a detergent (Triton X-100) prior to nuclease treatment. (i) Chip-based capillary electrophoresis (Bioanalyzer) showing that only miniscule amounts of dsDNA could be detected after EV lysis using a detergent prior to treatment with nucleases.

    Journal: Journal of Extracellular Vesicles

    Article Title: Large extracellular vesicles carry most of the tumour DNA circulating in prostate cancer patient plasma

    doi: 10.1080/20013078.2018.1505403

    Figure Lengend Snippet: Most extracellular DNA is packaged into L-EVs . (a) Tunable resistive pulse sensing (TRPS, qNano) using two different pore membranes (NP4000 and NP200) identified as L-EVs (left) and S-EVs (right) derived from PC3 cells. NP4000 membrane, which can detect particles with a diameter between 1.0 and 6.0 μm, was used for quantitation of L-EVs, while NP200 membrane, which can detect particles with a diameter between 60 and 400 nm, was used for quantitation of S-EVs. (b) Protein lysates from L-EVs and S-EVs purified by iodixanol density gradient (at 1.10 and 1.15 g/ml) were blotted with LO markers HSPA5 and CK18, and with Exo marker CD81. (c) Total DNA was quantified by Qubit Fluorometer in L-EVs and S-EVs isolated from PC3 and U87 cell lines. The plot shows the DNA ratio between L-EVs and S-EVs. (d) Double stranded (ds)DNA was quantified by High Sensitivity (HS) dsDNA Qubit Assay in L-EVs and S-EVs isolated from 1 ml of plasma from patients with mCRPC ( n = 40) and cancer-free individuals ( n = 6). (e) Quantification of both protein and DNA content in L-EVs and S-EVs isolated from conditioned media of 12.6 × 10 7 PC3 cells. (f) Single stranded (ss) and dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Exonuclease III, were quantified by Qubit. (g) Chip-based capillary electrophoresis (Bioanalyzer) showing the presence of dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Endonuclease III. L-EVs contain abundant DNA with a large peak around 10 kbp. Conversely, the amount of DNA in S-EVs is negligible. (h) ss- and dsDNA in PC3-derived L-EVs and S-EVs were quantified by Qubit after treatment with nucleases (DNase I and Exonuclease III) with or without addition of a detergent (Triton X-100) prior to nuclease treatment. (i) Chip-based capillary electrophoresis (Bioanalyzer) showing that only miniscule amounts of dsDNA could be detected after EV lysis using a detergent prior to treatment with nucleases.

    Article Snippet: Final library quality control was performed using the DNA High Sensitivity Qubit Kit (Invitrogen), the Bioanalyzer High Sensitivity Chip Kit (Agilent) and the 7900HT Fast qPCR machine (Applied Biosystems). qPCR was performed using the Illumina Universal Library Quantification Kit from KAPA Biosystems.

    Techniques: Tunable Resistive Pulse Sensing, Derivative Assay, Quantitation Assay, Purification, Marker, Isolation, HS DSDNA Qubit Assay, Chromatin Immunoprecipitation, Electrophoresis, Lysis

    Size preference of NGS and titration of transposase. Profiles of an NGS library are compared: Panel A shows what was loaded onto MiSeq (analyzed by Bioanalyzer) and Panel B shows what was actually sequenced. Short fragments are more efficient in forming clusters. Panel C shows profiles of the libraries generated with various ratios of transposon to DNA. Traces 1–8 represent tagmentation reactions of 4.44 ng of human DNA by 0.023 μL, 0.047 μL, 0.093 μL, 0.19 μL, 0.38 μL, 0.75 μL, 1.5 μL, and 3 μL of Mu transposon respectively (details in Materials and methods ). Reactions represented by Traces 6, 7, and 8 were under saturating conditions.

    Journal: PLoS ONE

    Article Title: New library construction method for single-cell genomes

    doi: 10.1371/journal.pone.0181163

    Figure Lengend Snippet: Size preference of NGS and titration of transposase. Profiles of an NGS library are compared: Panel A shows what was loaded onto MiSeq (analyzed by Bioanalyzer) and Panel B shows what was actually sequenced. Short fragments are more efficient in forming clusters. Panel C shows profiles of the libraries generated with various ratios of transposon to DNA. Traces 1–8 represent tagmentation reactions of 4.44 ng of human DNA by 0.023 μL, 0.047 μL, 0.093 μL, 0.19 μL, 0.38 μL, 0.75 μL, 1.5 μL, and 3 μL of Mu transposon respectively (details in Materials and methods ). Reactions represented by Traces 6, 7, and 8 were under saturating conditions.

    Article Snippet: Barcoded libraries were purified with AMPure beads and quantified based on the electropherogram obtained on the Agilent 2100 Bioanalyzer with Agilent High Sensitivity DNA Analysis Kit before they were sequenced on MiSeq™ using custom primers provided by the MuSeek Library Preparation Kit.

    Techniques: Next-Generation Sequencing, Titration, Generated

    Changes in the cfDNA concentration during systemic therapy (A) Change in the cfDNA concentration from baseline to first response assessment, according to the radiological response category. (B) Waterfall plot for the percentage change in the cfDNA concentration at first response assessment. (C) Change in the cfDNA concentration from baseline to the radiological best response, according to the radiological best response category. (D) Waterfall plot for the percentage change in the cfDNA concentration at assessment of radiological best response. cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer; PR, partial response; SD, stable disease; PD, progression of disease * Kruskal-Wallis test among PR, SD and PD groups ** Data are expressed as the median, followed by the interquartile range in parentheses *** Wilcoxon signed rank test between the cfDNA concentration at baseline and at first follow-up assessment or best response.

    Journal: Oncotarget

    Article Title: Quantification of circulating cell-free DNA to predict patient survival in non-small-cell lung cancer

    doi: 10.18632/oncotarget.21769

    Figure Lengend Snippet: Changes in the cfDNA concentration during systemic therapy (A) Change in the cfDNA concentration from baseline to first response assessment, according to the radiological response category. (B) Waterfall plot for the percentage change in the cfDNA concentration at first response assessment. (C) Change in the cfDNA concentration from baseline to the radiological best response, according to the radiological best response category. (D) Waterfall plot for the percentage change in the cfDNA concentration at assessment of radiological best response. cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer; PR, partial response; SD, stable disease; PD, progression of disease * Kruskal-Wallis test among PR, SD and PD groups ** Data are expressed as the median, followed by the interquartile range in parentheses *** Wilcoxon signed rank test between the cfDNA concentration at baseline and at first follow-up assessment or best response.

    Article Snippet: After cfDNA extraction, the purity of the cfDNA was measured with an Agilent High Sensitivity DNA kit and a Bioanalyzer 2100 instrument (Agilent Technologies, Santa Clara, CA, USA).

    Techniques: Concentration Assay

    Study flow diagram cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer, ADC, adenocarcinoma.

    Journal: Oncotarget

    Article Title: Quantification of circulating cell-free DNA to predict patient survival in non-small-cell lung cancer

    doi: 10.18632/oncotarget.21769

    Figure Lengend Snippet: Study flow diagram cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer, ADC, adenocarcinoma.

    Article Snippet: After cfDNA extraction, the purity of the cfDNA was measured with an Agilent High Sensitivity DNA kit and a Bioanalyzer 2100 instrument (Agilent Technologies, Santa Clara, CA, USA).

    Techniques: Flow Cytometry

    Kaplan-Meier estimates of PFS and OS according to the cfDNA concentration in patients with NSCLC (A) PFS and (B) OS according to the baseline cfDNA concentration (≤ 70 ng/mL vs. > 70 ng/mL) in all patients with NSCLC. (C) PFS and (D) OS according to the baseline cfDNA concentration (≤ 70 ng/mL vs. > 70 ng/mL) in chemo-naive patients with stage IV adenocarcinoma. cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer; PFS, progression-free survival; OS, overall survival.

    Journal: Oncotarget

    Article Title: Quantification of circulating cell-free DNA to predict patient survival in non-small-cell lung cancer

    doi: 10.18632/oncotarget.21769

    Figure Lengend Snippet: Kaplan-Meier estimates of PFS and OS according to the cfDNA concentration in patients with NSCLC (A) PFS and (B) OS according to the baseline cfDNA concentration (≤ 70 ng/mL vs. > 70 ng/mL) in all patients with NSCLC. (C) PFS and (D) OS according to the baseline cfDNA concentration (≤ 70 ng/mL vs. > 70 ng/mL) in chemo-naive patients with stage IV adenocarcinoma. cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer; PFS, progression-free survival; OS, overall survival.

    Article Snippet: After cfDNA extraction, the purity of the cfDNA was measured with an Agilent High Sensitivity DNA kit and a Bioanalyzer 2100 instrument (Agilent Technologies, Santa Clara, CA, USA).

    Techniques: Concentration Assay

    Circulating cfDNA time points coded by NSCLC patient identification number Graphical presentation of the association between the cfDNA level and the assessment of radiological response in patients with disease progression (A) and without progression (B) Change in the cfDNA concentration from baseline to best response, according to the radiological best response category; x-axis displays the time to clinical tumor progression. cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer; PR, partial response; SD, stable disease; PD, progression of disease * Kruskal-Wallis test among PR, SD and PD groups ** Data are expressed as the median, followed by the interquartile range in parentheses *** Wilcoxon signed rank test between the cfDNA concentration at baseline and at best response.

    Journal: Oncotarget

    Article Title: Quantification of circulating cell-free DNA to predict patient survival in non-small-cell lung cancer

    doi: 10.18632/oncotarget.21769

    Figure Lengend Snippet: Circulating cfDNA time points coded by NSCLC patient identification number Graphical presentation of the association between the cfDNA level and the assessment of radiological response in patients with disease progression (A) and without progression (B) Change in the cfDNA concentration from baseline to best response, according to the radiological best response category; x-axis displays the time to clinical tumor progression. cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer; PR, partial response; SD, stable disease; PD, progression of disease * Kruskal-Wallis test among PR, SD and PD groups ** Data are expressed as the median, followed by the interquartile range in parentheses *** Wilcoxon signed rank test between the cfDNA concentration at baseline and at best response.

    Article Snippet: After cfDNA extraction, the purity of the cfDNA was measured with an Agilent High Sensitivity DNA kit and a Bioanalyzer 2100 instrument (Agilent Technologies, Santa Clara, CA, USA).

    Techniques: Concentration Assay

    Circulating cfDNA kinetics in patients with NSCLCQuantitative cfDNA dynamics during treatment for NSCLC (A) Change in the cfDNA concentration from baseline to disease progression, according to the radiological best response category. (B) Change in the cfDNA concentration from the previous level to disease progression, according to the radiological best response category. Colors and symbols in the panel represent individual patient cfDNA kinetics; x-axis displays the time to clinical tumor progression. cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer; PR, partial response; SD, stable disease; PD, progression of disease * Kruskal-Wallis test among PR, SD and PD groups ** Data are expressed as the median, followed by the interquartile range in parentheses. *** Wilcoxon signed rank test between the cfDNA level at disease progression and the baseline or previous cfDNA level.

    Journal: Oncotarget

    Article Title: Quantification of circulating cell-free DNA to predict patient survival in non-small-cell lung cancer

    doi: 10.18632/oncotarget.21769

    Figure Lengend Snippet: Circulating cfDNA kinetics in patients with NSCLCQuantitative cfDNA dynamics during treatment for NSCLC (A) Change in the cfDNA concentration from baseline to disease progression, according to the radiological best response category. (B) Change in the cfDNA concentration from the previous level to disease progression, according to the radiological best response category. Colors and symbols in the panel represent individual patient cfDNA kinetics; x-axis displays the time to clinical tumor progression. cfDNA, cell-free DNA; NSCLC, non-small-cell lung cancer; PR, partial response; SD, stable disease; PD, progression of disease * Kruskal-Wallis test among PR, SD and PD groups ** Data are expressed as the median, followed by the interquartile range in parentheses. *** Wilcoxon signed rank test between the cfDNA level at disease progression and the baseline or previous cfDNA level.

    Article Snippet: After cfDNA extraction, the purity of the cfDNA was measured with an Agilent High Sensitivity DNA kit and a Bioanalyzer 2100 instrument (Agilent Technologies, Santa Clara, CA, USA).

    Techniques: Concentration Assay

    Application of Nextera DNA Flex to bacterial amplicons. a Libraries prepared using Nextera DNA Flex showed more consistent, even coverage compared with libraries prepared using Nextera XT; data depicts the sequence coverage of libraries prepared from the 3 kb E. coli amplicon. b PCR products ranging in size from 50 bp to 3 kb amplified from E. coli gDNA visualized on a 1% agarose gel. c Libraries prepared from a 1 ng input of these E. coli amplicons resulted in Bioanalyzer traces that depicted a slight increase in fragment size with increasing amplicon size. d Libraries were sequenced on a MiSeq and coverage of the E. coli genome determined for the different amplicon fragment size inputs. Sequenceable libraries were generated from amplicons ranging in size from 50 bp to 3 kb. e When sequencing data was downsampled to 25,000 reads, the larger fragment inputs were reaching a coverage maximum

    Journal: BMC Genomics

    Article Title: Bead-linked transposomes enable a normalization-free workflow for NGS library preparation

    doi: 10.1186/s12864-018-5096-9

    Figure Lengend Snippet: Application of Nextera DNA Flex to bacterial amplicons. a Libraries prepared using Nextera DNA Flex showed more consistent, even coverage compared with libraries prepared using Nextera XT; data depicts the sequence coverage of libraries prepared from the 3 kb E. coli amplicon. b PCR products ranging in size from 50 bp to 3 kb amplified from E. coli gDNA visualized on a 1% agarose gel. c Libraries prepared from a 1 ng input of these E. coli amplicons resulted in Bioanalyzer traces that depicted a slight increase in fragment size with increasing amplicon size. d Libraries were sequenced on a MiSeq and coverage of the E. coli genome determined for the different amplicon fragment size inputs. Sequenceable libraries were generated from amplicons ranging in size from 50 bp to 3 kb. e When sequencing data was downsampled to 25,000 reads, the larger fragment inputs were reaching a coverage maximum

    Article Snippet: Where described, library quality was determined by running 1 μl of the pooled library or an individual library on a Bioanalyzer (Agilent 2100 Bioanalyzer) using a High Sensitivity DNA kit (Agilent, cat. no. 5067–4626) or on a Fragment Analyzer (Advanced Analytical Fragment Analyzer) with the High Sensitivity NGS Fragment Analysis Kit (Advanced Analytical, cat. no. DNF-474).

    Techniques: Sequencing, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Generated

    Bioanalyzer traces of libraries prepared from various sample types and species. a Libraries prepared from samples with a varied degree of formalin fixation; a higher ΔCq indicates more FFPE-induced DNA degradation compared with a positive control. b Increasing FFPE-induced DNA degradation has a small effect on average fragment size but a marked effect on the total library yield. Increasing the DNA input from 100 ng to 150 ng did not increase library yield, indicating bead saturation at a DNA input of around 100 ng regardless of the degree of DNA degradation. c Libraries prepared from gDNA from a range of animal (human, Angus, and mouse), plant (Arabidopsis and alfalfa), and bacterial ( E. coli and B. cereus ) species

    Journal: BMC Genomics

    Article Title: Bead-linked transposomes enable a normalization-free workflow for NGS library preparation

    doi: 10.1186/s12864-018-5096-9

    Figure Lengend Snippet: Bioanalyzer traces of libraries prepared from various sample types and species. a Libraries prepared from samples with a varied degree of formalin fixation; a higher ΔCq indicates more FFPE-induced DNA degradation compared with a positive control. b Increasing FFPE-induced DNA degradation has a small effect on average fragment size but a marked effect on the total library yield. Increasing the DNA input from 100 ng to 150 ng did not increase library yield, indicating bead saturation at a DNA input of around 100 ng regardless of the degree of DNA degradation. c Libraries prepared from gDNA from a range of animal (human, Angus, and mouse), plant (Arabidopsis and alfalfa), and bacterial ( E. coli and B. cereus ) species

    Article Snippet: Where described, library quality was determined by running 1 μl of the pooled library or an individual library on a Bioanalyzer (Agilent 2100 Bioanalyzer) using a High Sensitivity DNA kit (Agilent, cat. no. 5067–4626) or on a Fragment Analyzer (Advanced Analytical Fragment Analyzer) with the High Sensitivity NGS Fragment Analysis Kit (Advanced Analytical, cat. no. DNF-474).

    Techniques: Formalin-fixed Paraffin-Embedded, Positive Control

    Application of Nextera DNA Flex to human amplicons. a Human leukocyte antigen (HLA) gene amplicons used as inputs for library preparation visualized on a 1% agarose gel. Lanes and expected amplicon sizes are as follows: 1, KBL Ladder; 2, HLA-A (4.1 kb); 3, HLA-B (2.8 kb); 4, HLA-C (4.2 kb); 5, HLA-DPA1 (10.3 kb); 6, HLA-DPB1 (9.7 kb); 7, HLA-DQA1 (7.3 kb); 8, HLA-DRB2 (4.6 kb); 9, HLA-DQB1 (7.1 kb). b Nextera DNA Flex library yields of all HLA amplicons were within the acceptable values of > 4 ng/μl and 9–13 ng/μl for 1 ng and 100–300 ng inputs, respectively. The yields for Nextera DNA Flex libraries were higher than for those prepared using TruSight HLA; for TruSight HLA, libraries were prepared from 1 ng of each amplicon and then pooled. c The Bioanalyzer profiles depict library fragment size distributions within the acceptable range; the distribution is narrower for the Nextera DNA Flex libraries (1 ng DNA inputs) than the TruSight HLA libraries. d Sequencing coverage depth and uniformity were higher for libraries prepared using Nextera DNA Flex (Flex) compared with TruSight HLA (TS HLA). e Libraries were sequenced on a NextSeq 550, with downsampling to 25,000 reads per amplicon. Library preparation using Nextera DNA Flex (orange) resulted in more uniform coverage of the entire human mitochondrial chromosome when compared with Nextera XT (grey). The location of the PCR primers used to create the two mtDNA amplicons are depicted by blue and red arrows. Dotted-line rectangle indicates the D-Loop region. f Zoomed in view shows more uniform coverage with Nextera DNA Flex within the D-Loop region

    Journal: BMC Genomics

    Article Title: Bead-linked transposomes enable a normalization-free workflow for NGS library preparation

    doi: 10.1186/s12864-018-5096-9

    Figure Lengend Snippet: Application of Nextera DNA Flex to human amplicons. a Human leukocyte antigen (HLA) gene amplicons used as inputs for library preparation visualized on a 1% agarose gel. Lanes and expected amplicon sizes are as follows: 1, KBL Ladder; 2, HLA-A (4.1 kb); 3, HLA-B (2.8 kb); 4, HLA-C (4.2 kb); 5, HLA-DPA1 (10.3 kb); 6, HLA-DPB1 (9.7 kb); 7, HLA-DQA1 (7.3 kb); 8, HLA-DRB2 (4.6 kb); 9, HLA-DQB1 (7.1 kb). b Nextera DNA Flex library yields of all HLA amplicons were within the acceptable values of > 4 ng/μl and 9–13 ng/μl for 1 ng and 100–300 ng inputs, respectively. The yields for Nextera DNA Flex libraries were higher than for those prepared using TruSight HLA; for TruSight HLA, libraries were prepared from 1 ng of each amplicon and then pooled. c The Bioanalyzer profiles depict library fragment size distributions within the acceptable range; the distribution is narrower for the Nextera DNA Flex libraries (1 ng DNA inputs) than the TruSight HLA libraries. d Sequencing coverage depth and uniformity were higher for libraries prepared using Nextera DNA Flex (Flex) compared with TruSight HLA (TS HLA). e Libraries were sequenced on a NextSeq 550, with downsampling to 25,000 reads per amplicon. Library preparation using Nextera DNA Flex (orange) resulted in more uniform coverage of the entire human mitochondrial chromosome when compared with Nextera XT (grey). The location of the PCR primers used to create the two mtDNA amplicons are depicted by blue and red arrows. Dotted-line rectangle indicates the D-Loop region. f Zoomed in view shows more uniform coverage with Nextera DNA Flex within the D-Loop region

    Article Snippet: Where described, library quality was determined by running 1 μl of the pooled library or an individual library on a Bioanalyzer (Agilent 2100 Bioanalyzer) using a High Sensitivity DNA kit (Agilent, cat. no. 5067–4626) or on a Fragment Analyzer (Advanced Analytical Fragment Analyzer) with the High Sensitivity NGS Fragment Analysis Kit (Advanced Analytical, cat. no. DNF-474).

    Techniques: Agarose Gel Electrophoresis, Amplification, Sequencing, Polymerase Chain Reaction

    Effect of blood sample storage (at 22°C) on exosome and exosome DNA concentration in plasma. A, Effect of blood storage on exosome concentration analyzed by NanoSight instrument under light scatter mode. Blood from each donor was divided into 4 aliquots and stored at 22°C. Plasma separated from each aliquot at indicated days, exosomes isolated and enumerated. B, Effect of blood storage on proteins CD9, CD63 and CD235a in plasma exosome pellet (isolated by Invitrogen method). C, Effect of storage on proteins CD41 and CD45 in plasma exosome pellet. D, Effect of blood storage on exosome DNA concentration as detected by β-actin ddPCR assay. Blood from each donor was divided into 4 aliquots and stored at 22°C. Plasma separated from each aliquot at indicated days, exosomes isolated. DNA extracted from exosomes and β-actin copy number detected by ddPCR assay. Error bars indicate SD. Panel B is in logarithmic scale.

    Journal: PLoS ONE

    Article Title: New evidence that a large proportion of human blood plasma cell-free DNA is localized in exosomes

    doi: 10.1371/journal.pone.0183915

    Figure Lengend Snippet: Effect of blood sample storage (at 22°C) on exosome and exosome DNA concentration in plasma. A, Effect of blood storage on exosome concentration analyzed by NanoSight instrument under light scatter mode. Blood from each donor was divided into 4 aliquots and stored at 22°C. Plasma separated from each aliquot at indicated days, exosomes isolated and enumerated. B, Effect of blood storage on proteins CD9, CD63 and CD235a in plasma exosome pellet (isolated by Invitrogen method). C, Effect of storage on proteins CD41 and CD45 in plasma exosome pellet. D, Effect of blood storage on exosome DNA concentration as detected by β-actin ddPCR assay. Blood from each donor was divided into 4 aliquots and stored at 22°C. Plasma separated from each aliquot at indicated days, exosomes isolated. DNA extracted from exosomes and β-actin copy number detected by ddPCR assay. Error bars indicate SD. Panel B is in logarithmic scale.

    Article Snippet: RNase treated or not treated exosome DNA was also analyzed by Agilent Bioanalyzer using Agilent High Sensitivity DNA kit. shows results from two donors.

    Techniques: Concentration Assay, Isolation

    Analysis of exosome DNA by agarose gel separation and Agilent Bioanalyzer. A, Exosome DNA without RNase treatment. B, Exosome DNA with RNase treatment. High molecular weight band is removed by RNase treatment indicating that band represents RNA. Low molecular weight band is resistant to RNase treatment indicating that it is DNA. Majority of exosome DNA are in 200 bp size range. C, Overlaid Agilent 2100 Bioanalyzer electropherograms. Exosome DNA was extracted from two individual donors. Exosome DNA from both donors were either treated with RNase or not treated. RNase treated and not treated DNA were analyzed by Agilent Bioanalyzer and RNase treated and not treated electropherograms were overlaid.

    Journal: PLoS ONE

    Article Title: New evidence that a large proportion of human blood plasma cell-free DNA is localized in exosomes

    doi: 10.1371/journal.pone.0183915

    Figure Lengend Snippet: Analysis of exosome DNA by agarose gel separation and Agilent Bioanalyzer. A, Exosome DNA without RNase treatment. B, Exosome DNA with RNase treatment. High molecular weight band is removed by RNase treatment indicating that band represents RNA. Low molecular weight band is resistant to RNase treatment indicating that it is DNA. Majority of exosome DNA are in 200 bp size range. C, Overlaid Agilent 2100 Bioanalyzer electropherograms. Exosome DNA was extracted from two individual donors. Exosome DNA from both donors were either treated with RNase or not treated. RNase treated and not treated DNA were analyzed by Agilent Bioanalyzer and RNase treated and not treated electropherograms were overlaid.

    Article Snippet: RNase treated or not treated exosome DNA was also analyzed by Agilent Bioanalyzer using Agilent High Sensitivity DNA kit. shows results from two donors.

    Techniques: Agarose Gel Electrophoresis, Molecular Weight

    Total and amplifiable DNA concentrations in plasma and plasma exosomes. A, Comparison of total DNA concentrations in plasma (median 6.86 ng/mL) and plasma exosomes (median 4.9 ng/mL). B, Comparison of total DNA concentrations in exosome pellet (median 5.6 ng/mL) and plasma supernatant (median 0.0 ng/mL). C, Comparison of β-actin DNA concentrations in plasma and plasma exosomes detected by a ddPCR assay. There was no statistically significant difference between β-actin DNA concentrations in plasma (median 1600 β-actin copies/mL plasma) and plasma exosomes (median 1560 β-actin copies/mL plasma). D, Comparison of β-actin DNA concentrations in plasma exosome pellet (median 888 β-actin copies/mL plasma) and plasma supernatant (median 52 β-actin copies/mL plasma) detected by ddPCR assay. The line inside of the box indicates median value. The limits of the box represent the 75th and 25th percentiles. The whiskers indicate the 10th and 90th percentiles. Panels A and C; n = 23. Panels B and D; n = 16. * p

    Journal: PLoS ONE

    Article Title: New evidence that a large proportion of human blood plasma cell-free DNA is localized in exosomes

    doi: 10.1371/journal.pone.0183915

    Figure Lengend Snippet: Total and amplifiable DNA concentrations in plasma and plasma exosomes. A, Comparison of total DNA concentrations in plasma (median 6.86 ng/mL) and plasma exosomes (median 4.9 ng/mL). B, Comparison of total DNA concentrations in exosome pellet (median 5.6 ng/mL) and plasma supernatant (median 0.0 ng/mL). C, Comparison of β-actin DNA concentrations in plasma and plasma exosomes detected by a ddPCR assay. There was no statistically significant difference between β-actin DNA concentrations in plasma (median 1600 β-actin copies/mL plasma) and plasma exosomes (median 1560 β-actin copies/mL plasma). D, Comparison of β-actin DNA concentrations in plasma exosome pellet (median 888 β-actin copies/mL plasma) and plasma supernatant (median 52 β-actin copies/mL plasma) detected by ddPCR assay. The line inside of the box indicates median value. The limits of the box represent the 75th and 25th percentiles. The whiskers indicate the 10th and 90th percentiles. Panels A and C; n = 23. Panels B and D; n = 16. * p

    Article Snippet: RNase treated or not treated exosome DNA was also analyzed by Agilent Bioanalyzer using Agilent High Sensitivity DNA kit. shows results from two donors.

    Techniques:

    Positive circulating tumor DNA (ctDNA) and short fragment size of plasma cell‐free DNA (cfDNA) were associated with poor prognosis. (Kaplan‐Meier method and log‐rank test). A,B, Prognosis was analyzed in 27 renal cell carcinoma (RCC) patients whose cfDNA samples were sequenced at pretreatment state. Association of ctDNA status (positive vs negative) for progression‐free survival (PFS) (A) and cancer‐specific survival (CSS) (B). C,D, Association of cfDNA fragment size using a microfluidics‐based platform between ≤166 bp (the prominent peak of the distribution of cfDNA fragments according to size) (short) and > 166 bp (long) for PFS (C) and CSS (D). E,F, Prognosis was analyzed in 13 RCC patients with metastasis whose cfDNA samples were sequenced at pretreatment state as in A‐D. Association of ctDNA status (positive vs negative) (E) and cfDNA fragment size (short vs long) (F) for CSS

    Journal: Cancer Science

    Article Title: Clinical significance of the mutational landscape and fragmentation of circulating tumor DNA in renal cell carcinoma, et al. Clinical significance of the mutational landscape and fragmentation of circulating tumor DNA in renal cell carcinoma

    doi: 10.1111/cas.13906

    Figure Lengend Snippet: Positive circulating tumor DNA (ctDNA) and short fragment size of plasma cell‐free DNA (cfDNA) were associated with poor prognosis. (Kaplan‐Meier method and log‐rank test). A,B, Prognosis was analyzed in 27 renal cell carcinoma (RCC) patients whose cfDNA samples were sequenced at pretreatment state. Association of ctDNA status (positive vs negative) for progression‐free survival (PFS) (A) and cancer‐specific survival (CSS) (B). C,D, Association of cfDNA fragment size using a microfluidics‐based platform between ≤166 bp (the prominent peak of the distribution of cfDNA fragments according to size) (short) and > 166 bp (long) for PFS (C) and CSS (D). E,F, Prognosis was analyzed in 13 RCC patients with metastasis whose cfDNA samples were sequenced at pretreatment state as in A‐D. Association of ctDNA status (positive vs negative) (E) and cfDNA fragment size (short vs long) (F) for CSS

    Article Snippet: 2.4 Global cfDNA concentration from 1 mL plasma was measured using the Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). cfDNA fragment size was measured using a microfluidics‐based platform, the Agilent 2100 Bioanalyzer with the High Sensitivity DNA Kit (Agilent Technologies, Santa Clara, CA, USA).

    Techniques:

    Renal cell carcinoma (RCC) patients with circulating tumor DNA (ctDNA) had shorter cell‐free DNA (cfDNA) fragments than those without ctDNA. A, Distributions of cfDNA fragments according to size were determined by targeted sequencing in 53 RCC patients. X‐axis shows cfDNA fragment size, and the Y‐axis shows the abundance of fragments of those specific sizes relative to the number of 166‐bp fragments. Red lines (n = 16) indicate the distributions of cfDNA fragments for patients with ctDNA, and blue lines (n = 37) for patients without ctDNA. B, Proportion of cfDNA fragments (PCF) was defined as the ratio of short cfDNA fragments (50‐166 bp; green) to long fragments (167‐250 bp; blue) as determined by next‐generation sequencing (NGS). Using a microfluidics‐based platform, average cfDNA fragment size in case 50 was classified as short (154 bp), whereas that in case 52 was classified as long (174 bp). C, PCF in patients with short cfDNA fragment size (≤166 bp, n = 24) as measured by a microfluidics‐based platform tended to be higher than in those with long cfDNA fragments ( > 166 bp, n = 29; P = .085). (Wilcoxon test). D, PCF was weakly correlated with cfDNA fragment size as determined by a microfluidics‐based platform (n = 53). (correlation analysis). E, Association between ctDNA status and cfDNA fragment size as determined by a microfluidics‐based platform (n = 53; P = .245). (Wilcoxon test). F, Association between ctDNA status and PCF (n = 53). * P

    Journal: Cancer Science

    Article Title: Clinical significance of the mutational landscape and fragmentation of circulating tumor DNA in renal cell carcinoma, et al. Clinical significance of the mutational landscape and fragmentation of circulating tumor DNA in renal cell carcinoma

    doi: 10.1111/cas.13906

    Figure Lengend Snippet: Renal cell carcinoma (RCC) patients with circulating tumor DNA (ctDNA) had shorter cell‐free DNA (cfDNA) fragments than those without ctDNA. A, Distributions of cfDNA fragments according to size were determined by targeted sequencing in 53 RCC patients. X‐axis shows cfDNA fragment size, and the Y‐axis shows the abundance of fragments of those specific sizes relative to the number of 166‐bp fragments. Red lines (n = 16) indicate the distributions of cfDNA fragments for patients with ctDNA, and blue lines (n = 37) for patients without ctDNA. B, Proportion of cfDNA fragments (PCF) was defined as the ratio of short cfDNA fragments (50‐166 bp; green) to long fragments (167‐250 bp; blue) as determined by next‐generation sequencing (NGS). Using a microfluidics‐based platform, average cfDNA fragment size in case 50 was classified as short (154 bp), whereas that in case 52 was classified as long (174 bp). C, PCF in patients with short cfDNA fragment size (≤166 bp, n = 24) as measured by a microfluidics‐based platform tended to be higher than in those with long cfDNA fragments ( > 166 bp, n = 29; P = .085). (Wilcoxon test). D, PCF was weakly correlated with cfDNA fragment size as determined by a microfluidics‐based platform (n = 53). (correlation analysis). E, Association between ctDNA status and cfDNA fragment size as determined by a microfluidics‐based platform (n = 53; P = .245). (Wilcoxon test). F, Association between ctDNA status and PCF (n = 53). * P

    Article Snippet: 2.4 Global cfDNA concentration from 1 mL plasma was measured using the Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). cfDNA fragment size was measured using a microfluidics‐based platform, the Agilent 2100 Bioanalyzer with the High Sensitivity DNA Kit (Agilent Technologies, Santa Clara, CA, USA).

    Techniques: Sequencing, Next-Generation Sequencing

    Clinical course monitoring in renal cell carcinoma (RCC) patients with circulating tumor DNA (ctDNA). Clinical course was analyzed using mutant allele frequency (MAF) of ctDNA by droplet digital PCR (ddPCR), and cell‐free DNA (cfDNA) fragment size by a microfluidics‐based platform in RCC patients with ctDNA. PD, progressive disease. A, In case 45, MAF of ctDNA was evaluated in TP53 and VHL . B, In case 53, MAF of ctDNA was evaluated in MTOR and TSC1

    Journal: Cancer Science

    Article Title: Clinical significance of the mutational landscape and fragmentation of circulating tumor DNA in renal cell carcinoma, et al. Clinical significance of the mutational landscape and fragmentation of circulating tumor DNA in renal cell carcinoma

    doi: 10.1111/cas.13906

    Figure Lengend Snippet: Clinical course monitoring in renal cell carcinoma (RCC) patients with circulating tumor DNA (ctDNA). Clinical course was analyzed using mutant allele frequency (MAF) of ctDNA by droplet digital PCR (ddPCR), and cell‐free DNA (cfDNA) fragment size by a microfluidics‐based platform in RCC patients with ctDNA. PD, progressive disease. A, In case 45, MAF of ctDNA was evaluated in TP53 and VHL . B, In case 53, MAF of ctDNA was evaluated in MTOR and TSC1

    Article Snippet: 2.4 Global cfDNA concentration from 1 mL plasma was measured using the Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). cfDNA fragment size was measured using a microfluidics‐based platform, the Agilent 2100 Bioanalyzer with the High Sensitivity DNA Kit (Agilent Technologies, Santa Clara, CA, USA).

    Techniques: Mutagenesis, Digital PCR

    EGF and FBS-induced EGR1 expression and co-recruitment of Pol2 with active kinases to EGR1, FOS, HBB and GAPDH loci . ( A ) HeLa S3 cells were treated with EGF (100 ng/ml) or 10% FBS and harvested at indicated time point then RNA extracted with Trizol followed by RT-qPCR measurements. EGR1 expression was normalized to RPLP0 mRNA ( n = 3;±SD). ( B ) Cells were treated as in A and collected at indicated time point. A total of 5 μg of whole cell extract was resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electrotransferred to PVDF membranes. Western blot analysis was performed using anti-pMEK1/2, anti-pERK1/2 and anti-β-Actin antibodies. ( C ) HeLa S3 cells were challenged with either EGF (100 ng/ml) or 10% FBS and fixed with 1% formaldehyde at indicated time point. Chromatin was isolated and used in ChIP assays with unspecific IgG, anti-Pol2, anti-pEGFR, anti-pMEK1/2 and anti-pERK1/2 antibody. Isolated DNA was used as the template in qPCR analyses with primers designed to amplify exon1 of EGR1 , exon1-intron1 junction of FOS gene and promoters of the β-globin ( HBB ), and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ). Data are expressed as a percentage of input chromatin ( n = 3;±SD).

    Journal: Nucleic Acids Research

    Article Title: Genome-wide co-localization of active EGFR and downstream ERK pathway kinases mirrors mitogen-inducible RNA polymerase 2 genomic occupancy

    doi: 10.1093/nar/gkw763

    Figure Lengend Snippet: EGF and FBS-induced EGR1 expression and co-recruitment of Pol2 with active kinases to EGR1, FOS, HBB and GAPDH loci . ( A ) HeLa S3 cells were treated with EGF (100 ng/ml) or 10% FBS and harvested at indicated time point then RNA extracted with Trizol followed by RT-qPCR measurements. EGR1 expression was normalized to RPLP0 mRNA ( n = 3;±SD). ( B ) Cells were treated as in A and collected at indicated time point. A total of 5 μg of whole cell extract was resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electrotransferred to PVDF membranes. Western blot analysis was performed using anti-pMEK1/2, anti-pERK1/2 and anti-β-Actin antibodies. ( C ) HeLa S3 cells were challenged with either EGF (100 ng/ml) or 10% FBS and fixed with 1% formaldehyde at indicated time point. Chromatin was isolated and used in ChIP assays with unspecific IgG, anti-Pol2, anti-pEGFR, anti-pMEK1/2 and anti-pERK1/2 antibody. Isolated DNA was used as the template in qPCR analyses with primers designed to amplify exon1 of EGR1 , exon1-intron1 junction of FOS gene and promoters of the β-globin ( HBB ), and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ). Data are expressed as a percentage of input chromatin ( n = 3;±SD).

    Article Snippet: The quality and quantity of all libraries were assessed on 2100 Bioanalyzer using High Sensitivity DNA Kit (Agilent Technologies) and by qPCR with Kapa Library Quantification Kit (KapaBiosystems), respectively.

    Techniques: Expressing, Quantitative RT-PCR, Polyacrylamide Gel Electrophoresis, Western Blot, Isolation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    EGF-inducible EGFR and ERK pathway components binding at ACTB and ACTG1 loci. ( A ) ChIP-seq overview of active kinases and Pol2 binding to ACTB and ACTG1 genes depicted in IGV genome browser. Pol2 ENCODE ChIP-Seq data for HeLa-S3 growing cells (ID: wgEncodeBroadHistoneHelas3Pol2bStdSig) were included to show similarities in transcriptional complex binding between datasets. A vertical dotted line marks TSS. ( B ) Time-course ChIP-qPCR measurements confirm EGFR and ERK pathway components binding to ACTB and ACTG1 following EGF treatment. ChIP analysis of sheared chromatin from a time course of EGF-treated (100 ng/ml EGF for 0, 10, 20, 45 and 60 min) HeLa-S3 cells was done using Matrix-ChIP method in 96-well polypropylene plates as described ( 8 ). qPCR was performed using primers (Supplementary Table S1) spanning an area depicted in a cartoon above each gene. ChIP data are expressed as DNA recovered as the percentage (%) of input DNA ( n = 3; mean ±SD). Statistical analysis of differences between mean DNA recovery for a given factor in quiescent cells and EGF time points was performed using t -tests. A P -value of ≤ 0.05 (*) was considered significant, **; P -value ≤ 0.01. ( C ) Time-course RT-qPCR analyses of ACTB and ACTG1 mRNA and pre-mRNA during EGF time course. Cells were harvested at indicated time point, RNA extracted with Trizol, DNAze treated and submitted to RT-qPCR measurements. RNA expression was normalized to RPLP0 mRNA and presented as fold change of expression measured in quiescent cells ( n = 3; mean ±SD). Statistical analysis of differences between mean cDNA levels for quiescent cells and given EGF time point was performed using t -tests. A P -value of ≤ 0.05 (*) was considered significant, **; P -value ≤ 0.01. Gene cartoon depicts localization of primers amplifying either mature (RT, spanning exon-exon) or unspliced (pre-mRNA, spanning exon-intron) form of transcript.

    Journal: Nucleic Acids Research

    Article Title: Genome-wide co-localization of active EGFR and downstream ERK pathway kinases mirrors mitogen-inducible RNA polymerase 2 genomic occupancy

    doi: 10.1093/nar/gkw763

    Figure Lengend Snippet: EGF-inducible EGFR and ERK pathway components binding at ACTB and ACTG1 loci. ( A ) ChIP-seq overview of active kinases and Pol2 binding to ACTB and ACTG1 genes depicted in IGV genome browser. Pol2 ENCODE ChIP-Seq data for HeLa-S3 growing cells (ID: wgEncodeBroadHistoneHelas3Pol2bStdSig) were included to show similarities in transcriptional complex binding between datasets. A vertical dotted line marks TSS. ( B ) Time-course ChIP-qPCR measurements confirm EGFR and ERK pathway components binding to ACTB and ACTG1 following EGF treatment. ChIP analysis of sheared chromatin from a time course of EGF-treated (100 ng/ml EGF for 0, 10, 20, 45 and 60 min) HeLa-S3 cells was done using Matrix-ChIP method in 96-well polypropylene plates as described ( 8 ). qPCR was performed using primers (Supplementary Table S1) spanning an area depicted in a cartoon above each gene. ChIP data are expressed as DNA recovered as the percentage (%) of input DNA ( n = 3; mean ±SD). Statistical analysis of differences between mean DNA recovery for a given factor in quiescent cells and EGF time points was performed using t -tests. A P -value of ≤ 0.05 (*) was considered significant, **; P -value ≤ 0.01. ( C ) Time-course RT-qPCR analyses of ACTB and ACTG1 mRNA and pre-mRNA during EGF time course. Cells were harvested at indicated time point, RNA extracted with Trizol, DNAze treated and submitted to RT-qPCR measurements. RNA expression was normalized to RPLP0 mRNA and presented as fold change of expression measured in quiescent cells ( n = 3; mean ±SD). Statistical analysis of differences between mean cDNA levels for quiescent cells and given EGF time point was performed using t -tests. A P -value of ≤ 0.05 (*) was considered significant, **; P -value ≤ 0.01. Gene cartoon depicts localization of primers amplifying either mature (RT, spanning exon-exon) or unspliced (pre-mRNA, spanning exon-intron) form of transcript.

    Article Snippet: The quality and quantity of all libraries were assessed on 2100 Bioanalyzer using High Sensitivity DNA Kit (Agilent Technologies) and by qPCR with Kapa Library Quantification Kit (KapaBiosystems), respectively.

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, RNA Expression, Expressing

    AP2M1 depletion reduces EGF-inducible Pol2, EGFR, MEK1/2 and ERK1/2 binding to EGR1 locus. ( A ) HeLa-S3 cells line were transfected with either one of two AP2M1 siRNAs or non-specific siRNA in a presence of Lipofectamine 3000. Twenty-four hours after transfection, cells were switched to serum free medium, and 48 h after quiescence, cells were treated with EGF (100 ng/ml) for 0, 45 and 180 min. Cells were harvested at indicated time point then RNA extracted with Trizol followed by RT-qPCR measurements. EGR1 and AP2M1 expression was normalized to RPLP0 mRNA ( n = 3; ±SD). (B ) Cells were prepared as in A and challenged with EGF (100 ng/ml) for 0, 5, 20 and 60 min then fixed, chromatin isolated and sheared. Matrix ChIP assay was done using antibodies to Pol2, pEGFR, pMEK1/2 and pERK1/2. ChIP data are expressed as DNA recovery in percentage (%) of input (means ± S.D., n = 3). Statistical analysis of differences between mean DNA recovery for control and AP2M1-depleted chromatin at given time point was performed using t -tests. ChIP results are shown for PCR product depicted on gene cartoon. A P -value of

    Journal: Nucleic Acids Research

    Article Title: Genome-wide co-localization of active EGFR and downstream ERK pathway kinases mirrors mitogen-inducible RNA polymerase 2 genomic occupancy

    doi: 10.1093/nar/gkw763

    Figure Lengend Snippet: AP2M1 depletion reduces EGF-inducible Pol2, EGFR, MEK1/2 and ERK1/2 binding to EGR1 locus. ( A ) HeLa-S3 cells line were transfected with either one of two AP2M1 siRNAs or non-specific siRNA in a presence of Lipofectamine 3000. Twenty-four hours after transfection, cells were switched to serum free medium, and 48 h after quiescence, cells were treated with EGF (100 ng/ml) for 0, 45 and 180 min. Cells were harvested at indicated time point then RNA extracted with Trizol followed by RT-qPCR measurements. EGR1 and AP2M1 expression was normalized to RPLP0 mRNA ( n = 3; ±SD). (B ) Cells were prepared as in A and challenged with EGF (100 ng/ml) for 0, 5, 20 and 60 min then fixed, chromatin isolated and sheared. Matrix ChIP assay was done using antibodies to Pol2, pEGFR, pMEK1/2 and pERK1/2. ChIP data are expressed as DNA recovery in percentage (%) of input (means ± S.D., n = 3). Statistical analysis of differences between mean DNA recovery for control and AP2M1-depleted chromatin at given time point was performed using t -tests. ChIP results are shown for PCR product depicted on gene cartoon. A P -value of

    Article Snippet: The quality and quantity of all libraries were assessed on 2100 Bioanalyzer using High Sensitivity DNA Kit (Agilent Technologies) and by qPCR with Kapa Library Quantification Kit (KapaBiosystems), respectively.

    Techniques: Binding Assay, Transfection, Quantitative RT-PCR, Expressing, Isolation, Chromatin Immunoprecipitation, Polymerase Chain Reaction

    Line charts of serum cfDNA, CEA and CYFRA21-1 concentrations at serial time-points in patients with NSCLC. SD, stable disease; PR, partial response; PD, progressive disease; cfDNA, cell-free DNA; NSCLC, non-small cell lung cancer. Disease was detected by medical imaging.

    Journal: Oncology Letters

    Article Title: A quantitative analysis of the potential biomarkers of non-small cell lung cancer by circulating cell-free DNA

    doi: 10.3892/ol.2018.9198

    Figure Lengend Snippet: Line charts of serum cfDNA, CEA and CYFRA21-1 concentrations at serial time-points in patients with NSCLC. SD, stable disease; PR, partial response; PD, progressive disease; cfDNA, cell-free DNA; NSCLC, non-small cell lung cancer. Disease was detected by medical imaging.

    Article Snippet: The presence of cfDNA and its fragment size distribution were evaluated by using the Agilent High Sensitivity DNA kit (Agilent Technologies, Inc., Santa Clara, CA, USA) on the 2100 Bioanalyzer.

    Techniques: Imaging

    Diagnostic utility of serum cfDNA, CEA and CYFRA21-1 in NSCLC patients. (A) ROC curves of serum cfDNA, CEA, CYFRA21-1 and the combination of the three markers for distinguishing NSCLC patients from normal controls. (B) ROC curves of pairwise combinations of cfDNA, CEA, and CYFRA21-1 for distinguishing NSCLC patients from normal controls. cfDNA, cell-free DNA; NSCLC, non-small cell lung cancer; ROC, receiver operating characteristic.

    Journal: Oncology Letters

    Article Title: A quantitative analysis of the potential biomarkers of non-small cell lung cancer by circulating cell-free DNA

    doi: 10.3892/ol.2018.9198

    Figure Lengend Snippet: Diagnostic utility of serum cfDNA, CEA and CYFRA21-1 in NSCLC patients. (A) ROC curves of serum cfDNA, CEA, CYFRA21-1 and the combination of the three markers for distinguishing NSCLC patients from normal controls. (B) ROC curves of pairwise combinations of cfDNA, CEA, and CYFRA21-1 for distinguishing NSCLC patients from normal controls. cfDNA, cell-free DNA; NSCLC, non-small cell lung cancer; ROC, receiver operating characteristic.

    Article Snippet: The presence of cfDNA and its fragment size distribution were evaluated by using the Agilent High Sensitivity DNA kit (Agilent Technologies, Inc., Santa Clara, CA, USA) on the 2100 Bioanalyzer.

    Techniques: Diagnostic Assay

    Overview of Quartz-Seq2 experimental processes. a Quartz-Seq2 consists of five steps. (1) Each single cell in a droplet is sorted into lysis buffer in each well of a 384-well PCR plate using flow cytometry analysis data. (2) Poly-adenylated RNA in each well is reverse-transcribed into first-strand cDNA with reverse transcription primer, which has a unique cell barcode ( CB ). We prepare 384 or 1536 kinds of cell barcode with a unique sequence based on the Sequence–Levenshtein distance (SeqLv). The edit distance of SeqLv is 5. The RT primer also has a UMI sequence for reduction of PCR bias (MB) and a poly(dT) sequence for binding to poly(A) RNA. (3) Cell barcode-labeled cDNAs from all 384 wells are promptly collected by centrifugation using assembled collectors. (4) Collected first-strand cDNAs are purified and concentrated for subsequent whole-transcript amplification. In the poly(A) tailing step, purified cDNA is extended with a poly(A) tail by terminal deoxynucleotidyl transferase ( TdT ). Subsequently, second-strand cDNA is synthesized with a tagging primer, which has a poly(dT) sequence. The resulting second-strand cDNA has a PCR primer sequence ( M ) at both ends of it. The cDNA is amplifiable in a subsequent PCR amplification. (5) For conversion from amplified cDNA to sequence library DNA, we fragment the amplified cDNA using the ultrasonicator Covaris. Such fragmented cDNA is ligated with a truncated Y-shaped sequence adaptor, which has an Illumina flow-cell binding sequence ( P7 ) and a pool barcode sequence ( PB ). The PB makes it possible to mix different sets of cell barcode-labeled cDNA. Ligated cDNA, which has CB and MB sequences, is enriched by PCR amplification. The resulting sequence library DNA contains P7 and P5 flow-cell binding sequences at respective ends of the DNA. We sequence the cell barcode site and the UMI site at Read1, the pool barcode site at Index1, and the transcript sequence at Read2. b The relationship between initial fastq reads and the number of single cells for sequence analysis in NextSeq500 runs. Typically, one sequence run with NextSeq 500/550 High Output v2 Kit reads out 400–450 M fastq reads. The x-axis represents the input cell number for one sequence run. The y-axis represents the initial data size (fastq reads) on average per cell. The red outline represents the typical range of shallow input read depth for a single cell. c We define the formula for calculating the UMI conversion efficiency. Each parameter is defined as follows: UMI sc is the number of UMI counts, assigned to a single-cell sample, fastq sc is the number of fastq reads derived from each single-cell sample, fastq non-sc is the number of fastq reads derived from non-single-cell samples, which include experimental byproducts such as WTA adaptors, WTA byproducts, and non-STAMPs. Initial fastq reads are composed of fastq sc and fastq non-sc

    Journal: Genome Biology

    Article Title: Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads

    doi: 10.1186/s13059-018-1407-3

    Figure Lengend Snippet: Overview of Quartz-Seq2 experimental processes. a Quartz-Seq2 consists of five steps. (1) Each single cell in a droplet is sorted into lysis buffer in each well of a 384-well PCR plate using flow cytometry analysis data. (2) Poly-adenylated RNA in each well is reverse-transcribed into first-strand cDNA with reverse transcription primer, which has a unique cell barcode ( CB ). We prepare 384 or 1536 kinds of cell barcode with a unique sequence based on the Sequence–Levenshtein distance (SeqLv). The edit distance of SeqLv is 5. The RT primer also has a UMI sequence for reduction of PCR bias (MB) and a poly(dT) sequence for binding to poly(A) RNA. (3) Cell barcode-labeled cDNAs from all 384 wells are promptly collected by centrifugation using assembled collectors. (4) Collected first-strand cDNAs are purified and concentrated for subsequent whole-transcript amplification. In the poly(A) tailing step, purified cDNA is extended with a poly(A) tail by terminal deoxynucleotidyl transferase ( TdT ). Subsequently, second-strand cDNA is synthesized with a tagging primer, which has a poly(dT) sequence. The resulting second-strand cDNA has a PCR primer sequence ( M ) at both ends of it. The cDNA is amplifiable in a subsequent PCR amplification. (5) For conversion from amplified cDNA to sequence library DNA, we fragment the amplified cDNA using the ultrasonicator Covaris. Such fragmented cDNA is ligated with a truncated Y-shaped sequence adaptor, which has an Illumina flow-cell binding sequence ( P7 ) and a pool barcode sequence ( PB ). The PB makes it possible to mix different sets of cell barcode-labeled cDNA. Ligated cDNA, which has CB and MB sequences, is enriched by PCR amplification. The resulting sequence library DNA contains P7 and P5 flow-cell binding sequences at respective ends of the DNA. We sequence the cell barcode site and the UMI site at Read1, the pool barcode site at Index1, and the transcript sequence at Read2. b The relationship between initial fastq reads and the number of single cells for sequence analysis in NextSeq500 runs. Typically, one sequence run with NextSeq 500/550 High Output v2 Kit reads out 400–450 M fastq reads. The x-axis represents the input cell number for one sequence run. The y-axis represents the initial data size (fastq reads) on average per cell. The red outline represents the typical range of shallow input read depth for a single cell. c We define the formula for calculating the UMI conversion efficiency. Each parameter is defined as follows: UMI sc is the number of UMI counts, assigned to a single-cell sample, fastq sc is the number of fastq reads derived from each single-cell sample, fastq non-sc is the number of fastq reads derived from non-single-cell samples, which include experimental byproducts such as WTA adaptors, WTA byproducts, and non-STAMPs. Initial fastq reads are composed of fastq sc and fastq non-sc

    Article Snippet: We checked the length distribution of amplified cDNA with an Agilent High Sensitivity DNA Kit (Agilent).

    Techniques: Lysis, Polymerase Chain Reaction, Flow Cytometry, Cytometry, Sequencing, Binding Assay, Labeling, Centrifugation, Purification, Amplification, Synthesized, Derivative Assay

    Sequence performance of Quartz-Seq2 with molecular biological improvements. a Improvement of poly(A) tagging efficiency. The relative DNA yield in various poly(A) tagging conditions using purified first-strand cDNA from 1 ng of total RNA. T55 buffer as the terminal deoxynucleotidyl transferase (TdT) buffer and the temperature condition “ Increment ” for the poly(A) tagging step improved the cDNA yield of whole-transcript amplification. Buffer compositions are indicated in Additional file 6 : Table S5. QuartzB represents use of a Quartz-Seq-like buffer as a positive control, in accordance with the approach described in the original Quartz-Seq paper. Finally, we quantified cDNA yield (300–9000 bp) and byproduct DNA yield (50–300 bp) using a Bioanalyzer (Agilent). The presented p value was obtained using two-tailed Welch’s t -test. b Reverse transcription efficiency with serially diluted RT enzymes. The x-axis represents the average relative RT qPCR score from ten genes. Detailed concentrations of RT enzymes are presented in Additional file 1 : Figure S7. c , f – h Comparison between Quartz-Seq2 in the RT25 condition and Quartz-Seq-like conditions regarding sequence performance. c We analyzed 384 wells with 10 pg of total RNA and used approximately 0.19 M fastq reads on average per well. We show the UMI count and gene count in box plots. d A scatter plot between the mean of gene expression and the variability of gene expression with 10 pg of total RNA in 384 wells. Red lines represent the theoretical variability of gene expression in the form of a Poisson distribution. e Gene expression reproducibility between bulk poly(A)-RNA-seq (1 μg of total RNA) and Quartz-Seq2 (10 pg of total RNA, averaged over 384 wells). f Dispersion of gene expression. The x-axis represents gene expression variability. g Reproducibility of gene expression for internal gene and external control RNA. h Accuracy of gene expression for internal gene and external control RNA

    Journal: Genome Biology

    Article Title: Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads

    doi: 10.1186/s13059-018-1407-3

    Figure Lengend Snippet: Sequence performance of Quartz-Seq2 with molecular biological improvements. a Improvement of poly(A) tagging efficiency. The relative DNA yield in various poly(A) tagging conditions using purified first-strand cDNA from 1 ng of total RNA. T55 buffer as the terminal deoxynucleotidyl transferase (TdT) buffer and the temperature condition “ Increment ” for the poly(A) tagging step improved the cDNA yield of whole-transcript amplification. Buffer compositions are indicated in Additional file 6 : Table S5. QuartzB represents use of a Quartz-Seq-like buffer as a positive control, in accordance with the approach described in the original Quartz-Seq paper. Finally, we quantified cDNA yield (300–9000 bp) and byproduct DNA yield (50–300 bp) using a Bioanalyzer (Agilent). The presented p value was obtained using two-tailed Welch’s t -test. b Reverse transcription efficiency with serially diluted RT enzymes. The x-axis represents the average relative RT qPCR score from ten genes. Detailed concentrations of RT enzymes are presented in Additional file 1 : Figure S7. c , f – h Comparison between Quartz-Seq2 in the RT25 condition and Quartz-Seq-like conditions regarding sequence performance. c We analyzed 384 wells with 10 pg of total RNA and used approximately 0.19 M fastq reads on average per well. We show the UMI count and gene count in box plots. d A scatter plot between the mean of gene expression and the variability of gene expression with 10 pg of total RNA in 384 wells. Red lines represent the theoretical variability of gene expression in the form of a Poisson distribution. e Gene expression reproducibility between bulk poly(A)-RNA-seq (1 μg of total RNA) and Quartz-Seq2 (10 pg of total RNA, averaged over 384 wells). f Dispersion of gene expression. The x-axis represents gene expression variability. g Reproducibility of gene expression for internal gene and external control RNA. h Accuracy of gene expression for internal gene and external control RNA

    Article Snippet: We checked the length distribution of amplified cDNA with an Agilent High Sensitivity DNA Kit (Agilent).

    Techniques: Sequencing, Purification, Amplification, Positive Control, Two Tailed Test, Quantitative RT-PCR, Expressing, RNA Sequencing Assay

    Association between EGFR L858R positivity and DNA amounts in regions 1 and 2 Peripheral blood was collected from 40 patients with NSCLC who carried EGFR L858R as verified by tissue biopsy and plasma DNA extracted by method 1000-A. DNA concentration and molarity were analyzed according to plasma L858R positivity as determined by the MPB-QP method. Concentration and molality of regions 1 and 2 DNA fragments were measured by an Agilent Bioanalyzer. The concentration ( A – C ) and molarity ( D – F ) of regions 1 and 2 are shown. Statistical analyses were performed with the Mann–Whitney U test. Two asterisks denote p

    Journal: Oncotarget

    Article Title: Automated DNA extraction using cellulose magnetic beads can improve EGFR point mutation detection with liquid biopsy by efficiently recovering short and long DNA fragments

    doi: 10.18632/oncotarget.25388

    Figure Lengend Snippet: Association between EGFR L858R positivity and DNA amounts in regions 1 and 2 Peripheral blood was collected from 40 patients with NSCLC who carried EGFR L858R as verified by tissue biopsy and plasma DNA extracted by method 1000-A. DNA concentration and molarity were analyzed according to plasma L858R positivity as determined by the MPB-QP method. Concentration and molality of regions 1 and 2 DNA fragments were measured by an Agilent Bioanalyzer. The concentration ( A – C ) and molarity ( D – F ) of regions 1 and 2 are shown. Statistical analyses were performed with the Mann–Whitney U test. Two asterisks denote p

    Article Snippet: We used the High Sensitivity DNA Kit (Agilent Technologies Inc., Santa Clara, CA, Product no. 5067–4626), a microchip, and analyzed it with the Agilent 2100 Bioanalyzer equipped with Expert 2100 software (Agilent Technologies Inc., Santa Clara, CA) according to the manufacturer's instructions.

    Techniques: Concentration Assay, MANN-WHITNEY

    Size distribution of plasma DNA analyzed by Agilent Bioanalyzer and its difference according to extraction method ( A ) Representative size distribution pattern with each plasma DNA extraction method. Blue shows 200-M, green is 200-A, and red is 1000-A. ( B ) The definitions of “Region 1” and “Region 2”. DNA concentration and molality were measured by an Agilent Bioanalyzer. Comparison among DNA isolation procedures (200-M, 200-A, and 1000-A) was performed for concentration ( C , D ) and molarity ( E , F ) with Freidman's test and multiple pairwise comparisons.

    Journal: Oncotarget

    Article Title: Automated DNA extraction using cellulose magnetic beads can improve EGFR point mutation detection with liquid biopsy by efficiently recovering short and long DNA fragments

    doi: 10.18632/oncotarget.25388

    Figure Lengend Snippet: Size distribution of plasma DNA analyzed by Agilent Bioanalyzer and its difference according to extraction method ( A ) Representative size distribution pattern with each plasma DNA extraction method. Blue shows 200-M, green is 200-A, and red is 1000-A. ( B ) The definitions of “Region 1” and “Region 2”. DNA concentration and molality were measured by an Agilent Bioanalyzer. Comparison among DNA isolation procedures (200-M, 200-A, and 1000-A) was performed for concentration ( C , D ) and molarity ( E , F ) with Freidman's test and multiple pairwise comparisons.

    Article Snippet: We used the High Sensitivity DNA Kit (Agilent Technologies Inc., Santa Clara, CA, Product no. 5067–4626), a microchip, and analyzed it with the Agilent 2100 Bioanalyzer equipped with Expert 2100 software (Agilent Technologies Inc., Santa Clara, CA) according to the manufacturer's instructions.

    Techniques: DNA Extraction, Concentration Assay

    DSBs enrichment workflow and specificity of the DNA DSBs ( A ) DSBs enrichment workflow by MTX treatment or Restriction endonuclease digestion for quality control. Fragments released from the streptavidin beads were amplified by PCR using sequencing primers and sequenced. ( B ) Quality control of in situ digestion and blunt-ending by capillary electrophoresis. The top two traces are for the endonuclease digestion and blunt-ending performed in liquid; the bottom two traces are in low melting point agarose gel. I represents the size of digestion product of a 567-bp fluorescence-labeled DNA fragment by restriction digestion while II shows the size of digestion product after blunt-ending; III and IV represent the above reactions respectively in low melting point agarose gel. The arrow in black represents the complete blunt-ending. X-axis represents the size of fragments(bp), Y-axis represents the detector signal of peak(rfu). ( C ) DSBs enrichment products separated by agarose gel electrophoresis indicated by white box. ( D–F ) Capillary electrophoresis to detect DSB enrichment products after SbfI ( D ), PmeI ( E ) and HindIII ( F ) digestion. TA clone sequencing confirmed the results. The arrow in red indicates the DSB enrichments on Capillary electrophoresis; the circle marked with red-dotted lines shows the restriction sites; the arrow in black shows the ligation point. X-axis represents the size of fragments(bp), while Y-axis represents the detector signal of peak(rfu). ( G, H ) Capillary electrophoresis to detect DSB enrichment products of normal mESCs cultured in complete medium ( G ) and cultured in complete medium with 0.12 μM MTX ( H ). The arrow in red indicates the DSB enrichments. X-axis represents the size of fragments(bp), while Y-axis represents the detector signal of peak(rfu).

    Journal: Nucleic Acids Research

    Article Title: Folate deficiency facilitates recruitment of upstream binding factor to hot spots of DNA double-strand breaks of rRNA genes and promotes its transcription

    doi: 10.1093/nar/gkw1208

    Figure Lengend Snippet: DSBs enrichment workflow and specificity of the DNA DSBs ( A ) DSBs enrichment workflow by MTX treatment or Restriction endonuclease digestion for quality control. Fragments released from the streptavidin beads were amplified by PCR using sequencing primers and sequenced. ( B ) Quality control of in situ digestion and blunt-ending by capillary electrophoresis. The top two traces are for the endonuclease digestion and blunt-ending performed in liquid; the bottom two traces are in low melting point agarose gel. I represents the size of digestion product of a 567-bp fluorescence-labeled DNA fragment by restriction digestion while II shows the size of digestion product after blunt-ending; III and IV represent the above reactions respectively in low melting point agarose gel. The arrow in black represents the complete blunt-ending. X-axis represents the size of fragments(bp), Y-axis represents the detector signal of peak(rfu). ( C ) DSBs enrichment products separated by agarose gel electrophoresis indicated by white box. ( D–F ) Capillary electrophoresis to detect DSB enrichment products after SbfI ( D ), PmeI ( E ) and HindIII ( F ) digestion. TA clone sequencing confirmed the results. The arrow in red indicates the DSB enrichments on Capillary electrophoresis; the circle marked with red-dotted lines shows the restriction sites; the arrow in black shows the ligation point. X-axis represents the size of fragments(bp), while Y-axis represents the detector signal of peak(rfu). ( G, H ) Capillary electrophoresis to detect DSB enrichment products of normal mESCs cultured in complete medium ( G ) and cultured in complete medium with 0.12 μM MTX ( H ). The arrow in red indicates the DSB enrichments. X-axis represents the size of fragments(bp), while Y-axis represents the detector signal of peak(rfu).

    Article Snippet: We assessed quality and quantity of DSB enrichment on a 2100 Bioanalyzer (Agilent) using a High Sensitivity DNA Kit (Agilent) and by qPCR using a Kapa Library Quantification Kit (Kapa Biosystems).

    Techniques: Amplification, Polymerase Chain Reaction, Sequencing, In Situ, Electrophoresis, Agarose Gel Electrophoresis, Fluorescence, Labeling, Ligation, Cell Culture

    ( A ) Microfluidic electrophoretic separation of the different methods of Y chromosome capture using a DNA high sensitivity (HS) Bioanalyzer assay. Sample 1: flow cytometry capture, Sample 2: laser capture microdissection, Sample 3: magnetic streptavidin-bead capture. The HS ladder (on left) ranges from 35 base pair (bp) to 7000 bp. All sample peaks appear between the lower and upper marker peaks (35–10380 bp). ( B ) Bioanalyzer high sensitivity profiles of each capture technique. The protocol for this assay is as follows: The captured DNA (putative Y chromosome) was sonicated to a size between 150 and 500 bp and after the sonication DNA was loaded on the Bioanalyzer assay. The sonication program (see 4.5.1. DNA Shearing) was tested previously to obtain the specific fragment size, which has been verified to be proper for preparing a DNA library.

    Journal: Scientific Reports

    Article Title: Methodology for Y Chromosome Capture: A complete genome sequence of  Y chromosome using flow cytometry, laser microdissection and magnetic streptavidin-beads

    doi: 10.1038/s41598-018-27819-x

    Figure Lengend Snippet: ( A ) Microfluidic electrophoretic separation of the different methods of Y chromosome capture using a DNA high sensitivity (HS) Bioanalyzer assay. Sample 1: flow cytometry capture, Sample 2: laser capture microdissection, Sample 3: magnetic streptavidin-bead capture. The HS ladder (on left) ranges from 35 base pair (bp) to 7000 bp. All sample peaks appear between the lower and upper marker peaks (35–10380 bp). ( B ) Bioanalyzer high sensitivity profiles of each capture technique. The protocol for this assay is as follows: The captured DNA (putative Y chromosome) was sonicated to a size between 150 and 500 bp and after the sonication DNA was loaded on the Bioanalyzer assay. The sonication program (see 4.5.1. DNA Shearing) was tested previously to obtain the specific fragment size, which has been verified to be proper for preparing a DNA library.

    Article Snippet: We checked the quality using the 2100 Bioanalyzer System with the Agilent Technologies High Sensitivity DNA Kit.

    Techniques: Flow Cytometry, Cytometry, Laser Capture Microdissection, Marker, Sonication

    Influence of anticoagulant and blood preservation conditions on quality of cfDNA from healthy volunteers cfDNA concentrations were examined at the indicated time after blood collection using sodium citrate tubes ( A ) or EDTA 2K tubes ( B ) from ten healthy volunteers. Blood storage temperature until plasma separation was 4° C (white box) or room temperature (gray box). Size distribution of plasma DNA was analyzed with an Agilent bioanalyzer® ; representative examples are shown in panels C-F. Sodium citrate tubes ( C , E ) or EDTA 2K tubes ( D , F ) were used for blood collection, and blood storage until plasma separation was at RT (C, D) or 4° C (E, F). DNA concentration of 1000 bp to 9000 bp fragments ( G ) and of 100 bp to 250 bp fragments ( H ) in all samples stored at 4° C was measured with an Agilent bioanalyzer® as described in “Materials and methods”. Blood was collected into sodium citrate tubes (white box) or EDTA 2K tubes (gray box). Statistical analyses were performed with Friedman’s rank test.

    Journal: Oncotarget

    Article Title: Investigation of appropriate pre-analytical procedure for circulating free DNA from liquid biopsy

    doi: 10.18632/oncotarget.25881

    Figure Lengend Snippet: Influence of anticoagulant and blood preservation conditions on quality of cfDNA from healthy volunteers cfDNA concentrations were examined at the indicated time after blood collection using sodium citrate tubes ( A ) or EDTA 2K tubes ( B ) from ten healthy volunteers. Blood storage temperature until plasma separation was 4° C (white box) or room temperature (gray box). Size distribution of plasma DNA was analyzed with an Agilent bioanalyzer® ; representative examples are shown in panels C-F. Sodium citrate tubes ( C , E ) or EDTA 2K tubes ( D , F ) were used for blood collection, and blood storage until plasma separation was at RT (C, D) or 4° C (E, F). DNA concentration of 1000 bp to 9000 bp fragments ( G ) and of 100 bp to 250 bp fragments ( H ) in all samples stored at 4° C was measured with an Agilent bioanalyzer® as described in “Materials and methods”. Blood was collected into sodium citrate tubes (white box) or EDTA 2K tubes (gray box). Statistical analyses were performed with Friedman’s rank test.

    Article Snippet: We used the High Sensitivity DNA Kit® (Agilent Technologies Inc., Santa Clara, CA, USA, Product no. 5067–4626), a microchip, and analyzed the result with an Agilent 2100 Bioanalyzer® equipped with Expert 2100 software (Agilent Technologies Inc., Santa Clara, CA, USA) according to the manufacturer’s instructions.

    Techniques: Preserving, Concentration Assay

    Influence of anticoagulant and blood preservation conditions on quality of cfDNA from healthy volunteers cfDNA concentrations were examined at the indicated time after blood collection using sodium citrate tubes ( A ) or EDTA 2K tubes ( B ) from ten healthy volunteers. Blood storage temperature until plasma separation was 4° C (white box) or room temperature (gray box). Size distribution of plasma DNA was analyzed with an Agilent bioanalyzer ® ; representative examples are shown in panels C-F. Sodium citrate tubes ( C , E ) or EDTA 2K tubes ( D , F ) were used for blood collection, and blood storage until plasma separation was at RT (C, D) or 4° C (E, F). DNA concentration of 1000 bp to 9000 bp fragments ( G ) and of 100 bp to 250 bp fragments ( H ) in all samples stored at 4° C was measured with an Agilent bioanalyzer ® as described in “Materials and methods”. Blood was collected into sodium citrate tubes (white box) or EDTA 2K tubes (gray box). Statistical analyses were performed with Friedman’s rank test.

    Journal: Oncotarget

    Article Title: Investigation of appropriate pre-analytical procedure for circulating free DNA from liquid biopsy

    doi: 10.18632/oncotarget.25881

    Figure Lengend Snippet: Influence of anticoagulant and blood preservation conditions on quality of cfDNA from healthy volunteers cfDNA concentrations were examined at the indicated time after blood collection using sodium citrate tubes ( A ) or EDTA 2K tubes ( B ) from ten healthy volunteers. Blood storage temperature until plasma separation was 4° C (white box) or room temperature (gray box). Size distribution of plasma DNA was analyzed with an Agilent bioanalyzer ® ; representative examples are shown in panels C-F. Sodium citrate tubes ( C , E ) or EDTA 2K tubes ( D , F ) were used for blood collection, and blood storage until plasma separation was at RT (C, D) or 4° C (E, F). DNA concentration of 1000 bp to 9000 bp fragments ( G ) and of 100 bp to 250 bp fragments ( H ) in all samples stored at 4° C was measured with an Agilent bioanalyzer ® as described in “Materials and methods”. Blood was collected into sodium citrate tubes (white box) or EDTA 2K tubes (gray box). Statistical analyses were performed with Friedman’s rank test.

    Article Snippet: We used the High Sensitivity DNA Kit® (Agilent Technologies Inc., Santa Clara, CA, USA, Product no. 5067–4626), a microchip, and analyzed the result with an Agilent 2100 Bioanalyzer® equipped with Expert 2100 software (Agilent Technologies Inc., Santa Clara, CA, USA) according to the manufacturer’s instructions.

    Techniques: Preserving, Concentration Assay

    In Agilent Bioanalyzer gel-like image of cDNA. This image shows produced cDNA samples quality controls. After library construction for the quality of the libraries was validated using the Agilent High Sensitivity DNA kit on the Agilent 2100 Bioanalyzer. DNA ladder (L), Lanes 1-3-5 (control cDNA library), the lanes 3-5 are ten-fold diluted sample 1. Lanels 2-4-6 (Multiple myeloma cDNA library). The lanes 4-6 are ten- fold diluted sample 2. Lane 7 (negative). Green lines indicate the low weight (35 base pairs) DNA ladder, Purple lines the high weight (10380 base pairs) DNA ladder.

    Journal: Balkan Medical Journal

    Article Title: Investigation of Gene Expressions of Myeloma Cells in the Bone Marrow of Multiple Myeloma Patients by Transcriptome Analysis

    doi: 10.4274/balkanmedj.2018.0356

    Figure Lengend Snippet: In Agilent Bioanalyzer gel-like image of cDNA. This image shows produced cDNA samples quality controls. After library construction for the quality of the libraries was validated using the Agilent High Sensitivity DNA kit on the Agilent 2100 Bioanalyzer. DNA ladder (L), Lanes 1-3-5 (control cDNA library), the lanes 3-5 are ten-fold diluted sample 1. Lanels 2-4-6 (Multiple myeloma cDNA library). The lanes 4-6 are ten- fold diluted sample 2. Lane 7 (negative). Green lines indicate the low weight (35 base pairs) DNA ladder, Purple lines the high weight (10380 base pairs) DNA ladder.

    Article Snippet: The quality of the libraries was validated using the Agilent High Sensitivity DNA kit on the Agilent 2100 Bioanalyzer ( ).

    Techniques: Produced, cDNA Library Assay

    L-EVs isolated from plasma of mCRPC patients contain large size DNA with tumour aberrations . (a) DNA quantitation in plasma-derived L-EVs and S-EVs, as well as EV-free DNA obtained from mCRPC patients ( n = 4) indicates that a significant portion of circulating DNA is enclosed in L-EVs. The EV-free DNA was extracted from EV-depleted plasma. (b) Chip-based capillary electrophoresis (Bioanalyzer) showing that L-EVs isolated from 1 ml of mCRPC patient plasma ( n = 3) contain high-quality, large size DNA. (c) EVs were isolated from 1 ml of plasma obtained from mCRPC patients ( n = 4), EV DNA was extracted in agarose plugs by incubation in lysis buffer for 48 h, and high molecular weight DNA was resolved by PFGE. Similar to L-EVs in vitro , patient plasma-derived L-EVs contain high molecular weight DNA (100 kbp–2 Mbp) (indicated by red dashed lines). (d) MYC/PTEN copy number imbalance in PC3 L-EVs was analysed by digital PCR (dPCR) using different amounts of DNA template. (e) MYC/PTEN copy number imbalance was evaluated in L-EVs isolated from 1 ml of mCRPC patient plasma ( n = 6) and compared to MYC/PTEN copy number in normal DNA extracted from the indicated benign cell lines. * p

    Journal: Journal of Extracellular Vesicles

    Article Title: Large extracellular vesicles carry most of the tumour DNA circulating in prostate cancer patient plasma

    doi: 10.1080/20013078.2018.1505403

    Figure Lengend Snippet: L-EVs isolated from plasma of mCRPC patients contain large size DNA with tumour aberrations . (a) DNA quantitation in plasma-derived L-EVs and S-EVs, as well as EV-free DNA obtained from mCRPC patients ( n = 4) indicates that a significant portion of circulating DNA is enclosed in L-EVs. The EV-free DNA was extracted from EV-depleted plasma. (b) Chip-based capillary electrophoresis (Bioanalyzer) showing that L-EVs isolated from 1 ml of mCRPC patient plasma ( n = 3) contain high-quality, large size DNA. (c) EVs were isolated from 1 ml of plasma obtained from mCRPC patients ( n = 4), EV DNA was extracted in agarose plugs by incubation in lysis buffer for 48 h, and high molecular weight DNA was resolved by PFGE. Similar to L-EVs in vitro , patient plasma-derived L-EVs contain high molecular weight DNA (100 kbp–2 Mbp) (indicated by red dashed lines). (d) MYC/PTEN copy number imbalance in PC3 L-EVs was analysed by digital PCR (dPCR) using different amounts of DNA template. (e) MYC/PTEN copy number imbalance was evaluated in L-EVs isolated from 1 ml of mCRPC patient plasma ( n = 6) and compared to MYC/PTEN copy number in normal DNA extracted from the indicated benign cell lines. * p

    Article Snippet: DNA quality was assessed using the Bioanalyzer DNA High Sensitivity Chip Kit (Agilent).

    Techniques: Isolation, Quantitation Assay, Derivative Assay, Chromatin Immunoprecipitation, Electrophoresis, Incubation, Lysis, Molecular Weight, In Vitro, Digital PCR

    Most extracellular DNA is packaged into L-EVs . (a) Tunable resistive pulse sensing (TRPS, qNano) using two different pore membranes (NP4000 and NP200) identified as L-EVs (left) and S-EVs (right) derived from PC3 cells. NP4000 membrane, which can detect particles with a diameter between 1.0 and 6.0 μm, was used for quantitation of L-EVs, while NP200 membrane, which can detect particles with a diameter between 60 and 400 nm, was used for quantitation of S-EVs. (b) Protein lysates from L-EVs and S-EVs purified by iodixanol density gradient (at 1.10 and 1.15 g/ml) were blotted with LO markers HSPA5 and CK18, and with Exo marker CD81. (c) Total DNA was quantified by Qubit Fluorometer in L-EVs and S-EVs isolated from PC3 and U87 cell lines. The plot shows the DNA ratio between L-EVs and S-EVs. (d) Double stranded (ds)DNA was quantified by High Sensitivity (HS) dsDNA Qubit Assay in L-EVs and S-EVs isolated from 1 ml of plasma from patients with mCRPC ( n = 40) and cancer-free individuals ( n = 6). (e) Quantification of both protein and DNA content in L-EVs and S-EVs isolated from conditioned media of 12.6 × 10 7 PC3 cells. (f) Single stranded (ss) and dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Exonuclease III, were quantified by Qubit. (g) Chip-based capillary electrophoresis (Bioanalyzer) showing the presence of dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Endonuclease III. L-EVs contain abundant DNA with a large peak around 10 kbp. Conversely, the amount of DNA in S-EVs is negligible. (h) ss- and dsDNA in PC3-derived L-EVs and S-EVs were quantified by Qubit after treatment with nucleases (DNase I and Exonuclease III) with or without addition of a detergent (Triton X-100) prior to nuclease treatment. (i) Chip-based capillary electrophoresis (Bioanalyzer) showing that only miniscule amounts of dsDNA could be detected after EV lysis using a detergent prior to treatment with nucleases.

    Journal: Journal of Extracellular Vesicles

    Article Title: Large extracellular vesicles carry most of the tumour DNA circulating in prostate cancer patient plasma

    doi: 10.1080/20013078.2018.1505403

    Figure Lengend Snippet: Most extracellular DNA is packaged into L-EVs . (a) Tunable resistive pulse sensing (TRPS, qNano) using two different pore membranes (NP4000 and NP200) identified as L-EVs (left) and S-EVs (right) derived from PC3 cells. NP4000 membrane, which can detect particles with a diameter between 1.0 and 6.0 μm, was used for quantitation of L-EVs, while NP200 membrane, which can detect particles with a diameter between 60 and 400 nm, was used for quantitation of S-EVs. (b) Protein lysates from L-EVs and S-EVs purified by iodixanol density gradient (at 1.10 and 1.15 g/ml) were blotted with LO markers HSPA5 and CK18, and with Exo marker CD81. (c) Total DNA was quantified by Qubit Fluorometer in L-EVs and S-EVs isolated from PC3 and U87 cell lines. The plot shows the DNA ratio between L-EVs and S-EVs. (d) Double stranded (ds)DNA was quantified by High Sensitivity (HS) dsDNA Qubit Assay in L-EVs and S-EVs isolated from 1 ml of plasma from patients with mCRPC ( n = 40) and cancer-free individuals ( n = 6). (e) Quantification of both protein and DNA content in L-EVs and S-EVs isolated from conditioned media of 12.6 × 10 7 PC3 cells. (f) Single stranded (ss) and dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Exonuclease III, were quantified by Qubit. (g) Chip-based capillary electrophoresis (Bioanalyzer) showing the presence of dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Endonuclease III. L-EVs contain abundant DNA with a large peak around 10 kbp. Conversely, the amount of DNA in S-EVs is negligible. (h) ss- and dsDNA in PC3-derived L-EVs and S-EVs were quantified by Qubit after treatment with nucleases (DNase I and Exonuclease III) with or without addition of a detergent (Triton X-100) prior to nuclease treatment. (i) Chip-based capillary electrophoresis (Bioanalyzer) showing that only miniscule amounts of dsDNA could be detected after EV lysis using a detergent prior to treatment with nucleases.

    Article Snippet: DNA quality was assessed using the Bioanalyzer DNA High Sensitivity Chip Kit (Agilent).

    Techniques: Tunable Resistive Pulse Sensing, Derivative Assay, Quantitation Assay, Purification, Marker, Isolation, HS DSDNA Qubit Assay, Chromatin Immunoprecipitation, Electrophoresis, Lysis

    Analysis of plasma cfDNA obtained from one representative pregnant donor blood stored at 22°C using Agilent Bioanalyzer 2100 instrument and Agilent DNA high sensitivity Kit. A, Overlaid electropherograms of plasma cfDNA extracted from blood stored (at 22°C) in K 3 EDTA tubes at days 0, 3, 7, 14 and 28. B, Overlaid electropherograms of plasma cfDNA extracted from blood stored (at 22°C) in ProTeck tubes at days 0, 3, 7, 14 and 28. C, Bioanalyzer gel image for blood stored in K 3 EDTA tubes. D, Bioanalyzer gel image for blood stored in ProTeck tubes. Cell-free DNA obtained from 3 pregnant donors were analyzed. This figure shows only the results of one representative pregnant donor.

    Journal: PLoS ONE

    Article Title: A novel approach to stabilize fetal cell-free DNA fraction in maternal blood samples for extended period of time

    doi: 10.1371/journal.pone.0208508

    Figure Lengend Snippet: Analysis of plasma cfDNA obtained from one representative pregnant donor blood stored at 22°C using Agilent Bioanalyzer 2100 instrument and Agilent DNA high sensitivity Kit. A, Overlaid electropherograms of plasma cfDNA extracted from blood stored (at 22°C) in K 3 EDTA tubes at days 0, 3, 7, 14 and 28. B, Overlaid electropherograms of plasma cfDNA extracted from blood stored (at 22°C) in ProTeck tubes at days 0, 3, 7, 14 and 28. C, Bioanalyzer gel image for blood stored in K 3 EDTA tubes. D, Bioanalyzer gel image for blood stored in ProTeck tubes. Cell-free DNA obtained from 3 pregnant donors were analyzed. This figure shows only the results of one representative pregnant donor.

    Article Snippet: Concentrated maternal cfDNA was analyzed by Agilent Bioanalyzer 2100 instrument and Agilent DNA High Sensitivity Kit following manufacturer’s recommended protocol.

    Techniques:

    Analysis of plasma cfDNA obtained from one representative pregnant donor blood stored at 30°C using Agilent Bioanalyzer 2100 instrument and Agilent DNA high sensitivity Kit. A, Overlaid electropherograms of plasma cfDNA extracted from blood stored in K 3 EDTA tubes at days 0, 2, 3, 7 and 14. B, Overlaid electropherograms of plasma cfDNA extracted from blood stored in ProTeck tubes at days 0, 2, 3, 7 and 14. C, Bioanalyzer gel image for blood stored in K 3 EDTA tubes. D, Bioanalyzer gel image for blood stored in ProTeck tubes. Cell-free DNA obtained from 3 pregnant donors were analyzed. This figure shows only the results of one representative pregnant donor.

    Journal: PLoS ONE

    Article Title: A novel approach to stabilize fetal cell-free DNA fraction in maternal blood samples for extended period of time

    doi: 10.1371/journal.pone.0208508

    Figure Lengend Snippet: Analysis of plasma cfDNA obtained from one representative pregnant donor blood stored at 30°C using Agilent Bioanalyzer 2100 instrument and Agilent DNA high sensitivity Kit. A, Overlaid electropherograms of plasma cfDNA extracted from blood stored in K 3 EDTA tubes at days 0, 2, 3, 7 and 14. B, Overlaid electropherograms of plasma cfDNA extracted from blood stored in ProTeck tubes at days 0, 2, 3, 7 and 14. C, Bioanalyzer gel image for blood stored in K 3 EDTA tubes. D, Bioanalyzer gel image for blood stored in ProTeck tubes. Cell-free DNA obtained from 3 pregnant donors were analyzed. This figure shows only the results of one representative pregnant donor.

    Article Snippet: Concentrated maternal cfDNA was analyzed by Agilent Bioanalyzer 2100 instrument and Agilent DNA High Sensitivity Kit following manufacturer’s recommended protocol.

    Techniques:

    Analysis of plasma cfDNA obtained from one representative pregnant donor blood stored at 4°C using Agilent Bioanalyzer 2100 instrument and Agilent DNA high sensitivity Kit. A, Overlaid electropherograms of plasma cfDNA extracted from blood stored in K 3 EDTA tubes at days 0, 3, 7, 14 and 21. B, Overlaid electropherograms of plasma cfDNA extracted from blood stored in ProTeck tubes at days 0, 3, 7, 14 and 21. C, Bioanalyzer gel image for blood stored in K 3 EDTA tubes. D, Bioanalyzer gel image for blood stored in ProTeck tubes. Cell-free DNA obtained from 3 pregnant donors were analyzed. This figure shows only the results of one representative pregnant donor.

    Journal: PLoS ONE

    Article Title: A novel approach to stabilize fetal cell-free DNA fraction in maternal blood samples for extended period of time

    doi: 10.1371/journal.pone.0208508

    Figure Lengend Snippet: Analysis of plasma cfDNA obtained from one representative pregnant donor blood stored at 4°C using Agilent Bioanalyzer 2100 instrument and Agilent DNA high sensitivity Kit. A, Overlaid electropherograms of plasma cfDNA extracted from blood stored in K 3 EDTA tubes at days 0, 3, 7, 14 and 21. B, Overlaid electropherograms of plasma cfDNA extracted from blood stored in ProTeck tubes at days 0, 3, 7, 14 and 21. C, Bioanalyzer gel image for blood stored in K 3 EDTA tubes. D, Bioanalyzer gel image for blood stored in ProTeck tubes. Cell-free DNA obtained from 3 pregnant donors were analyzed. This figure shows only the results of one representative pregnant donor.

    Article Snippet: Concentrated maternal cfDNA was analyzed by Agilent Bioanalyzer 2100 instrument and Agilent DNA High Sensitivity Kit following manufacturer’s recommended protocol.

    Techniques:

    NEXSON enhances the reproducibility of chromatin shearing. ( A ) Size distribution after chromatin shearing when extracting nuclei with either NEXSON or chemical and Dounce treatment from a single, formaldehyde-fixed cell batch (human monocytes). Chromatin was sheared with identical sonicator settings and DNA size distribution was analyzed using capillary electrophoresis. Note that differences in ultrasound exposure between the two workflows (NEXSON or chemical + Dounce) were normalized prior chromatin shearing. Electropherograms, generated with Agilent expert 2100 software, show the size distribution of the respective samples. Optimal chromatin size distribution for ChIP-seq is located between the two bars (100–800 bp). x axis: base pairs (bp), y axis: fluorescence units (FU). ( B ) Quantitative analysis of the chromatin size distribution of three different samples after nuclei extraction with NEXSON or chemical + Dounce homogenizer treatment. Bars show the percentage of DNA fragments in the optimal (between 100 and 800 bp, white bars) or inadequate (800–10 000 bp, gray bars) size range for ChIP-seq. Percentages of fragments in the respective region are calculated with Agilent Bioanalyzer software and averaged (error bars indicate s.d.; n = 3: fixed monocytes, IMR-90 and hepatocytes samples).

    Journal: Nucleic Acids Research

    Article Title: Standardizing chromatin research: a simple and universal method for ChIP-seq

    doi: 10.1093/nar/gkv1495

    Figure Lengend Snippet: NEXSON enhances the reproducibility of chromatin shearing. ( A ) Size distribution after chromatin shearing when extracting nuclei with either NEXSON or chemical and Dounce treatment from a single, formaldehyde-fixed cell batch (human monocytes). Chromatin was sheared with identical sonicator settings and DNA size distribution was analyzed using capillary electrophoresis. Note that differences in ultrasound exposure between the two workflows (NEXSON or chemical + Dounce) were normalized prior chromatin shearing. Electropherograms, generated with Agilent expert 2100 software, show the size distribution of the respective samples. Optimal chromatin size distribution for ChIP-seq is located between the two bars (100–800 bp). x axis: base pairs (bp), y axis: fluorescence units (FU). ( B ) Quantitative analysis of the chromatin size distribution of three different samples after nuclei extraction with NEXSON or chemical + Dounce homogenizer treatment. Bars show the percentage of DNA fragments in the optimal (between 100 and 800 bp, white bars) or inadequate (800–10 000 bp, gray bars) size range for ChIP-seq. Percentages of fragments in the respective region are calculated with Agilent Bioanalyzer software and averaged (error bars indicate s.d.; n = 3: fixed monocytes, IMR-90 and hepatocytes samples).

    Article Snippet: Fragment size distribution was analyzed by capillary electrophoresis (Agilent 2100 Bioanalyzer) using the High Sensitivity DNA ChIP kit (Agilent, 5067–4626).

    Techniques: Electrophoresis, Generated, Software, Chromatin Immunoprecipitation, Fluorescence