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Agilent technologies high sensitivity dna kit
Changes in the <t>cfDNA</t> 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 <t>DNA;</t> 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.
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1) Product Images from "Quantification of circulating cell-free DNA to predict patient survival in non-small-cell lung cancer"

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

Journal: Oncotarget

doi: 10.18632/oncotarget.21769

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.
Figure Legend 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.

Techniques Used: Concentration Assay

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

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: Concentration Assay

2) Product Images from "Bead-linked transposomes enable a normalization-free workflow for NGS library preparation"

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

Journal: BMC Genomics

doi: 10.1186/s12864-018-5096-9

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

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

3) Product Images from "New evidence that a large proportion of human blood plasma cell-free DNA is localized in exosomes"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0183915

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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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
Figure Legend 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

Techniques Used:

4) Product Images from "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"

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

Journal: Cancer Science

doi: 10.1111/cas.13906

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
Figure Legend 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

Techniques Used:

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: Mutagenesis, Digital PCR

5) Product Images from "Genome-wide co-localization of active EGFR and downstream ERK pathway kinases mirrors mitogen-inducible RNA polymerase 2 genomic occupancy"

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

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw763

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).
Figure Legend 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).

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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
Figure Legend 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

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

6) Product Images from "A quantitative analysis of the potential biomarkers of non-small cell lung cancer by circulating cell-free DNA"

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

Journal: Oncology Letters

doi: 10.3892/ol.2018.9198

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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: Diagnostic Assay

7) Product Images from "Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads"

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

Journal: Genome Biology

doi: 10.1186/s13059-018-1407-3

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

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

8) Product Images from "Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads"

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

Journal: Genome Biology

doi: 10.1186/s13059-018-1407-3

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
Figure Legend 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

Techniques Used: 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 : 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 : 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
Figure Legend 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 : 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 : 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

Techniques Used: Sequencing, Purification, Positive Control, Two Tailed Test, Expressing, RNA Sequencing Assay

9) Product Images from "Ecophylogeny of the endospheric root fungal microbiome of co-occurring Agrostis stolonifera"

Article Title: Ecophylogeny of the endospheric root fungal microbiome of co-occurring Agrostis stolonifera

Journal: PeerJ

doi: 10.7717/peerj.3454

Phylogenetic tree of the Basidiomycota related root fungal microbiome OTUs. ML tree based on 432 bp of SSU rRNA gene sequences amplified from roots of Agrostis stolonifera . The tree was constructed using representative sequences of the OTUs (taxa without names) and the closest reference sequences (taxa names in italic) from the non-redundant SILVA SSURef ARB database (release 115). Barplots represent the mean expression ratio for each OTU among all samples. Null values indicate that this OTU was not detected in the RNA fraction, value = 1 indicates that this OTU was not detected in the DNA fraction, value = 0.5 indicates that the sum of the relative abundance between DNA and RNA fractions was equal. Green bars: values below 0.5, red bars: values ≥ 0.5. Error bars indicate ±SE. Grey circles indicate the relative abundance of each OTU in the whole dataset. Node support values above 50 are given in the following order: bootstrap values and Bayesian posterior probabilities.
Figure Legend Snippet: Phylogenetic tree of the Basidiomycota related root fungal microbiome OTUs. ML tree based on 432 bp of SSU rRNA gene sequences amplified from roots of Agrostis stolonifera . The tree was constructed using representative sequences of the OTUs (taxa without names) and the closest reference sequences (taxa names in italic) from the non-redundant SILVA SSURef ARB database (release 115). Barplots represent the mean expression ratio for each OTU among all samples. Null values indicate that this OTU was not detected in the RNA fraction, value = 1 indicates that this OTU was not detected in the DNA fraction, value = 0.5 indicates that the sum of the relative abundance between DNA and RNA fractions was equal. Green bars: values below 0.5, red bars: values ≥ 0.5. Error bars indicate ±SE. Grey circles indicate the relative abundance of each OTU in the whole dataset. Node support values above 50 are given in the following order: bootstrap values and Bayesian posterior probabilities.

Techniques Used: Amplification, Construct, Expressing

Phylogenetic tree of the core microbiome OTUs at the level of 19 co-occurring Agrostis stolonifera plants. Tree construction was based on maximum likelihood. Only bootstrap values above 50 are indicated. The tree was constructed using sequences representative of the OTUs (taxa without names) and the closest reference sequences (taxa names in italic) from the non-redundant SILVA SSURef ARB database (release 115). ‘*’: taxa belonging to the ‘DNA core’, ‘ †’: taxa belonging to the ‘RNA core’. Stacked bars indicate the mean relative abundance of each taxon in the DNA (blue) and RNA (red) fractions of the 19 samples. Some taxa belonging to the ‘DNA core’ are also found in the RNA fractions but not in all samples and reciprocally.
Figure Legend Snippet: Phylogenetic tree of the core microbiome OTUs at the level of 19 co-occurring Agrostis stolonifera plants. Tree construction was based on maximum likelihood. Only bootstrap values above 50 are indicated. The tree was constructed using sequences representative of the OTUs (taxa without names) and the closest reference sequences (taxa names in italic) from the non-redundant SILVA SSURef ARB database (release 115). ‘*’: taxa belonging to the ‘DNA core’, ‘ †’: taxa belonging to the ‘RNA core’. Stacked bars indicate the mean relative abundance of each taxon in the DNA (blue) and RNA (red) fractions of the 19 samples. Some taxa belonging to the ‘DNA core’ are also found in the RNA fractions but not in all samples and reciprocally.

Techniques Used: Construct

Alpha diversity and taxonomic composition of fungal communities in DNA and RNA fractions. (A) Distribution of Hill diversity numbers in DNA and RNA sequence data analyses. Different commonly used diversity indexes are special cases of Hill numbers (e.g., q = 0 corresponds to the species richness S, q = 1 corresponds to the exponential of the Shannon-Wiener diversity index, q = 2 corresponds to the inverse Simpson index). Asterisks indicate significantly different means between DNA and RNA fractions (paired t -tests). ‘ns’: p > 0.05, ‘*’: p
Figure Legend Snippet: Alpha diversity and taxonomic composition of fungal communities in DNA and RNA fractions. (A) Distribution of Hill diversity numbers in DNA and RNA sequence data analyses. Different commonly used diversity indexes are special cases of Hill numbers (e.g., q = 0 corresponds to the species richness S, q = 1 corresponds to the exponential of the Shannon-Wiener diversity index, q = 2 corresponds to the inverse Simpson index). Asterisks indicate significantly different means between DNA and RNA fractions (paired t -tests). ‘ns’: p > 0.05, ‘*’: p

Techniques Used: Sequencing

10) Product Images from "Automated DNA extraction using cellulose magnetic beads can improve EGFR point mutation detection with liquid biopsy by efficiently recovering short and long DNA fragments"

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

Journal: Oncotarget

doi: 10.18632/oncotarget.25388

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
Figure Legend 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

Techniques Used: 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.
Figure Legend 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.

Techniques Used: DNA Extraction, Concentration Assay

11) Product Images from "Automated DNA extraction using cellulose magnetic beads can improve EGFR point mutation detection with liquid biopsy by efficiently recovering short and long DNA fragments"

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

Journal: Oncotarget

doi: 10.18632/oncotarget.25388

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
Figure Legend 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

Techniques Used: 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.
Figure Legend 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.

Techniques Used: DNA Extraction, Concentration Assay

12) Product Images from "Generating Exome Enriched Sequencing Libraries from Formalin-Fixed, Paraffin-Embedded Tissue DNA for Next Generation Sequencing"

Article Title: Generating Exome Enriched Sequencing Libraries from Formalin-Fixed, Paraffin-Embedded Tissue DNA for Next Generation Sequencing

Journal: Current protocols in human genetics

doi: 10.1002/cphg.27

DNA fragmentation quality check. 50ng of FFPE derived DNA was sheared using the Covaris E220 instrument. Following clean up each sample was diluted 1:5 and run on a 2100 BioAnalyzer High Sensitivity DNA chip to check the DNA fragment size distribution and sample concentration. A broad distribution of sizes should be expected between 100-2000bp is typical, with the majority of the fragments in the 200-800bp range.
Figure Legend Snippet: DNA fragmentation quality check. 50ng of FFPE derived DNA was sheared using the Covaris E220 instrument. Following clean up each sample was diluted 1:5 and run on a 2100 BioAnalyzer High Sensitivity DNA chip to check the DNA fragment size distribution and sample concentration. A broad distribution of sizes should be expected between 100-2000bp is typical, with the majority of the fragments in the 200-800bp range.

Techniques Used: Formalin-fixed Paraffin-Embedded, Derivative Assay, Chromatin Immunoprecipitation, Concentration Assay

Pre-Capture amplification quality check. Adapter ligated libraries were amplified prior to hybridization capture. Following clean up each sample was diluted 1:100 and run on a 2100 BioAnalyzer High Sensitivity DNA chip to confirm library fragment size and concentration. A focused size distribution between 200-800bp is typical. Quality of FFPE samples may affect how this distribution appears (average size between 250-550bp). Fragment size includes the added length of bases (123bp) from the ligated adapters.
Figure Legend Snippet: Pre-Capture amplification quality check. Adapter ligated libraries were amplified prior to hybridization capture. Following clean up each sample was diluted 1:100 and run on a 2100 BioAnalyzer High Sensitivity DNA chip to confirm library fragment size and concentration. A focused size distribution between 200-800bp is typical. Quality of FFPE samples may affect how this distribution appears (average size between 250-550bp). Fragment size includes the added length of bases (123bp) from the ligated adapters.

Techniques Used: Amplification, Hybridization, Chromatin Immunoprecipitation, Concentration Assay, Formalin-fixed Paraffin-Embedded

13) Product Images from "Generating Exome Enriched Sequencing Libraries from Formalin-Fixed, Paraffin-Embedded Tissue DNA for Next Generation Sequencing"

Article Title: Generating Exome Enriched Sequencing Libraries from Formalin-Fixed, Paraffin-Embedded Tissue DNA for Next Generation Sequencing

Journal: Current protocols in human genetics

doi: 10.1002/cphg.27

DNA fragmentation quality check. 50ng of FFPE derived DNA was sheared using the Covaris E220 instrument. Following clean up each sample was diluted 1:5 and run on a 2100 BioAnalyzer High Sensitivity DNA chip to check the DNA fragment size distribution and sample concentration. A broad distribution of sizes should be expected between 100-2000bp is typical, with the majority of the fragments in the 200-800bp range.
Figure Legend Snippet: DNA fragmentation quality check. 50ng of FFPE derived DNA was sheared using the Covaris E220 instrument. Following clean up each sample was diluted 1:5 and run on a 2100 BioAnalyzer High Sensitivity DNA chip to check the DNA fragment size distribution and sample concentration. A broad distribution of sizes should be expected between 100-2000bp is typical, with the majority of the fragments in the 200-800bp range.

Techniques Used: Formalin-fixed Paraffin-Embedded, Derivative Assay, Chromatin Immunoprecipitation, Concentration Assay

Pre-Capture amplification quality check. Adapter ligated libraries were amplified prior to hybridization capture. Following clean up each sample was diluted 1:100 and run on a 2100 BioAnalyzer High Sensitivity DNA chip to confirm library fragment size and concentration. A focused size distribution between 200-800bp is typical. Quality of FFPE samples may affect how this distribution appears (average size between 250-550bp). Fragment size includes the added length of bases (123bp) from the ligated adapters.
Figure Legend Snippet: Pre-Capture amplification quality check. Adapter ligated libraries were amplified prior to hybridization capture. Following clean up each sample was diluted 1:100 and run on a 2100 BioAnalyzer High Sensitivity DNA chip to confirm library fragment size and concentration. A focused size distribution between 200-800bp is typical. Quality of FFPE samples may affect how this distribution appears (average size between 250-550bp). Fragment size includes the added length of bases (123bp) from the ligated adapters.

Techniques Used: Amplification, Hybridization, Chromatin Immunoprecipitation, Concentration Assay, Formalin-fixed Paraffin-Embedded

14) Product Images from "Investigation of Gene Expressions of Myeloma Cells in the Bone Marrow of Multiple Myeloma Patients by Transcriptome Analysis"

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

Journal: Balkan Medical Journal

doi: 10.4274/balkanmedj.2018.0356

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.
Figure Legend 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.

Techniques Used: Produced, cDNA Library Assay

15) Product Images from "Adapting the Smart-seq2 Protocol for Robust Single Worm RNA-seq"

Article Title: Adapting the Smart-seq2 Protocol for Robust Single Worm RNA-seq

Journal: Bio-protocol

doi: 10.21769/BioProtoc.2729

Overview of the Smart-seq2 protocol adapted for individual nematodes . 1. A nematode is cut and lysed to release total RNA. 2. The Oligo(dT) binds the poly(A) tail at the 3′ end of mRNA (section A of Procedure). 3. The 1st strand of cDNA is synthesized by MMLV reverse transcriptase (RT) which adds non-template guided cytosines at the 3′ end of the cDNA. These cytosines are used to anchor the LNA-modified TSO. Reverse transcriptase then uses the TSO as a template to complete the 1st strand (section B of Procedure). 4. The ISPCR primer anneals to the 3′ end of the 1st strand allowing DNA polymerase to bind and to synthesize the 2nd strand of cDNA and subsequently amplify the cDNA (section C of Procedure). 5. The double-stranded (ds) cDNA is purified and checked for quality (sections D and E of Procedure). 6. Transposomes tagment the ds cDNA by fragmenting it and ligating adapter sequences (section F of Procedure). 7. The tagmented ds cDNA is cleaned and 8. Ligated to sample specific indexes (section G of Procedure).
Figure Legend Snippet: Overview of the Smart-seq2 protocol adapted for individual nematodes . 1. A nematode is cut and lysed to release total RNA. 2. The Oligo(dT) binds the poly(A) tail at the 3′ end of mRNA (section A of Procedure). 3. The 1st strand of cDNA is synthesized by MMLV reverse transcriptase (RT) which adds non-template guided cytosines at the 3′ end of the cDNA. These cytosines are used to anchor the LNA-modified TSO. Reverse transcriptase then uses the TSO as a template to complete the 1st strand (section B of Procedure). 4. The ISPCR primer anneals to the 3′ end of the 1st strand allowing DNA polymerase to bind and to synthesize the 2nd strand of cDNA and subsequently amplify the cDNA (section C of Procedure). 5. The double-stranded (ds) cDNA is purified and checked for quality (sections D and E of Procedure). 6. Transposomes tagment the ds cDNA by fragmenting it and ligating adapter sequences (section F of Procedure). 7. The tagmented ds cDNA is cleaned and 8. Ligated to sample specific indexes (section G of Procedure).

Techniques Used: Synthesized, Modification, Purification

16) Product Images from "Genome-wide mapping of embedded ribonucleotides and other non-canonical nucleotides using emRiboSeq and EndoSeq"

Article Title: Genome-wide mapping of embedded ribonucleotides and other non-canonical nucleotides using emRiboSeq and EndoSeq

Journal: Nature protocols

doi: 10.1038/nprot.2015.099

Library quality control and anticipated results. ( a ) Sonicated DNA separated by agarose gel electrophoresis (Step 25) shows an average fragment size of approximately 400 bp. ( b ) Bioanalyzer result (Step 84) for an emRiboSeq library shows a typical trace (left) and gel-like image (right) with a peak for fragments between ˜180 and ˜300 bp in size (black bar). Standards (green and purple bars) of defined size and amount allow quantification. FU, arbitrary fluorescence units. ( c ) Agarose gel electrophoresis of PCR products after 15, 16 and 17 cycles of amplification (Steps 81-83) of the same library shows product between 200 and 300 bp in size. ( d ) Sequencing results for libraries generated using Nb.BtsI are highly reproducibility between different strains (POL, wildtype polymerase; pol1-L868M, increased Pol-α ribonucleotide incorporation) after normalizing read counts to sequence tags per million (TPM). The majority of bona fide Nb.BtsI sites were present at maximal frequency, although some sites were present at lower frequencies. This is the result of partial loss during size selection because of their close proximity to other cleavage sites, a highly reproducible finding between independent libraries (Spearman's rho=0.82, p
Figure Legend Snippet: Library quality control and anticipated results. ( a ) Sonicated DNA separated by agarose gel electrophoresis (Step 25) shows an average fragment size of approximately 400 bp. ( b ) Bioanalyzer result (Step 84) for an emRiboSeq library shows a typical trace (left) and gel-like image (right) with a peak for fragments between ˜180 and ˜300 bp in size (black bar). Standards (green and purple bars) of defined size and amount allow quantification. FU, arbitrary fluorescence units. ( c ) Agarose gel electrophoresis of PCR products after 15, 16 and 17 cycles of amplification (Steps 81-83) of the same library shows product between 200 and 300 bp in size. ( d ) Sequencing results for libraries generated using Nb.BtsI are highly reproducibility between different strains (POL, wildtype polymerase; pol1-L868M, increased Pol-α ribonucleotide incorporation) after normalizing read counts to sequence tags per million (TPM). The majority of bona fide Nb.BtsI sites were present at maximal frequency, although some sites were present at lower frequencies. This is the result of partial loss during size selection because of their close proximity to other cleavage sites, a highly reproducible finding between independent libraries (Spearman's rho=0.82, p

Techniques Used: Sonication, Agarose Gel Electrophoresis, Fluorescence, Polymerase Chain Reaction, Amplification, Sequencing, Generated, Selection

17) Product Images from "Folate deficiency facilitates recruitment of upstream binding factor to hot spots of DNA double-strand breaks of rRNA genes and promotes its transcription"

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

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw1208

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).
Figure Legend 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).

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

18) Product Images from "Investigation of appropriate pre-analytical procedure for circulating free DNA from liquid biopsy"

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

Journal: Oncotarget

doi: 10.18632/oncotarget.25881

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.
Figure Legend 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.

Techniques Used: Preserving, Concentration Assay

19) Product Images from "Investigation of appropriate pre-analytical procedure for circulating free DNA from liquid biopsy"

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

Journal: Oncotarget

doi: 10.18632/oncotarget.25881

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.
Figure Legend 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.

Techniques Used: Preserving, Concentration Assay

20) Product Images from "New evidence that a large proportion of human blood plasma cell-free DNA is localized in exosomes"

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0183915

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.
Figure Legend 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.

Techniques Used: Agarose Gel Electrophoresis, Molecular Weight

21) Product Images from "Investigation of Gene Expressions of Myeloma Cells in the Bone Marrow of Multiple Myeloma Patients by Transcriptome Analysis"

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

Journal: Balkan Medical Journal

doi: 10.4274/balkanmedj.2018.0356

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.
Figure Legend 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.

Techniques Used: Produced, cDNA Library Assay

22) Product Images from "Exosomes maintain cellular homeostasis by excreting harmful DNA from cells"

Article Title: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells

Journal: Nature Communications

doi: 10.1038/ncomms15287

Exosomes contain chromosome fragments. ( a ) transmission electron microscopy micrograph of MVE in pre-senescent TIG-3 cells following immuno-gold labelling for dsDNA. Gold particles are depicted as black dots. Right image shows a digitally zoomed area of exosome. ( b , c ) Exosomal DNA isolated from pre-senescent TIG-3 cells were subjected to size distribution analysis using Electrophoresis Bioanalyzer system ( b ) or to deep sequencing analysis ( c ). Genomic DNA of TIG-3 cells are also subjected to deep sequencing analysis, as control ( c ). The read count of each 500-kb bin was normalized to RPKM and corrected by the mappability ( c ). ( d ) Purified exosomes from pre-senescent TIG-3 cells were subjected to sucrose density-gradient separation followed by western blotting using antibodies shown right, NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles and quantitative PCR analysis for detection of genomic DNA fragments (GRM7 and GPC6).
Figure Legend Snippet: Exosomes contain chromosome fragments. ( a ) transmission electron microscopy micrograph of MVE in pre-senescent TIG-3 cells following immuno-gold labelling for dsDNA. Gold particles are depicted as black dots. Right image shows a digitally zoomed area of exosome. ( b , c ) Exosomal DNA isolated from pre-senescent TIG-3 cells were subjected to size distribution analysis using Electrophoresis Bioanalyzer system ( b ) or to deep sequencing analysis ( c ). Genomic DNA of TIG-3 cells are also subjected to deep sequencing analysis, as control ( c ). The read count of each 500-kb bin was normalized to RPKM and corrected by the mappability ( c ). ( d ) Purified exosomes from pre-senescent TIG-3 cells were subjected to sucrose density-gradient separation followed by western blotting using antibodies shown right, NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles and quantitative PCR analysis for detection of genomic DNA fragments (GRM7 and GPC6).

Techniques Used: Transmission Assay, Electron Microscopy, Isolation, Electrophoresis, Sequencing, Purification, Western Blot, Real-time Polymerase Chain Reaction

23) Product Images from "Optimization of Extraction of Circulating RNAs from Plasma – Enabling Small RNA Sequencing"

Article Title: Optimization of Extraction of Circulating RNAs from Plasma – Enabling Small RNA Sequencing

Journal: PLoS ONE

doi: 10.1371/journal.pone.0107259

High Sensitivity DNA Bioanalyzer assay as checkpoint for correct size selection during library preparation. All nine samples showed adaptor/RNA/adaptor-constructs in appropriate sizes. One electropherogram is shown as representative example. The lengths of adaptor-ligated constructs from all nine samples were reported as indicated in the column peak size [bp] . The initial peak at 35 bp and the final peak at 10.380 bp are marker peaks that are system inherent included in all runs.
Figure Legend Snippet: High Sensitivity DNA Bioanalyzer assay as checkpoint for correct size selection during library preparation. All nine samples showed adaptor/RNA/adaptor-constructs in appropriate sizes. One electropherogram is shown as representative example. The lengths of adaptor-ligated constructs from all nine samples were reported as indicated in the column peak size [bp] . The initial peak at 35 bp and the final peak at 10.380 bp are marker peaks that are system inherent included in all runs.

Techniques Used: Selection, Construct, Marker

24) Product Images from "Integrative microRNA and mRNA deep-sequencing expression profiling in endemic Burkitt lymphoma"

Article Title: Integrative microRNA and mRNA deep-sequencing expression profiling in endemic Burkitt lymphoma

Journal: BMC Cancer

doi: 10.1186/s12885-017-3711-9

Aberrant transcriptome expression pivotal to eBL lymphomagenesis. a Schematic illustration of the aberrant gene expression and miRNA mediated regulatory changes that would initiate lymphomagenesis as a result of DNA damage. Combined loss of p53 function due to small interfering RNA-mediated regulation of ATM and NLK together with upregulation of TFAP4, would facilitate survival of cells with the c-myc-Igh chromosomal translocation and MYC induced cell cycle progression initiating eBL tumor development. ATM checkpoint kinase, transduces genomic stress signals to halt cell cycle progression in response to DNA damage. It is critical in the regulation of apoptosis and lymphomagenesis in c-myc induced lymphomas. ATM is downregulated in eBL and it is targeted by 4 miRs that are Upregulated in eBL. NLK is required for the upregulation of P53 expression in response to DNA damage. It interacts with P53 to enhance its stability and activity by abrogating MDM2 mediated degradation. NLK is downregulated in eBL tumor cells and also targeted by 2 miRs that are upregulated in eBL tumor cells. TFAP4/AP4 is a central mediator of cell cycle progression in response to c-MYC activation. b RNA seq. Expression counts of MYC , TFAP4 , ATM and NLK in eBL tumor cells and GC B cells. c Hierarchical clustering of eBL and GC B cells based on the expression profiles of MYC , TFAP4 , ATM and NLK also revealed a clear separation of the two groups. d . miRNA seq. Expression counts of hsa-miR-26a-5p, hsa-miR-27b-3p, hsa-miR-30b-5p, miR-17~92-cluster members (hsa-miR-19b-3p, and hsa-miR-92a-3p), and let-7-family miRs (hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7d-5p, hsa-let-7e-5p, and hsa-let-7 g-5p) in eBL tumor cells and GC B cells
Figure Legend Snippet: Aberrant transcriptome expression pivotal to eBL lymphomagenesis. a Schematic illustration of the aberrant gene expression and miRNA mediated regulatory changes that would initiate lymphomagenesis as a result of DNA damage. Combined loss of p53 function due to small interfering RNA-mediated regulation of ATM and NLK together with upregulation of TFAP4, would facilitate survival of cells with the c-myc-Igh chromosomal translocation and MYC induced cell cycle progression initiating eBL tumor development. ATM checkpoint kinase, transduces genomic stress signals to halt cell cycle progression in response to DNA damage. It is critical in the regulation of apoptosis and lymphomagenesis in c-myc induced lymphomas. ATM is downregulated in eBL and it is targeted by 4 miRs that are Upregulated in eBL. NLK is required for the upregulation of P53 expression in response to DNA damage. It interacts with P53 to enhance its stability and activity by abrogating MDM2 mediated degradation. NLK is downregulated in eBL tumor cells and also targeted by 2 miRs that are upregulated in eBL tumor cells. TFAP4/AP4 is a central mediator of cell cycle progression in response to c-MYC activation. b RNA seq. Expression counts of MYC , TFAP4 , ATM and NLK in eBL tumor cells and GC B cells. c Hierarchical clustering of eBL and GC B cells based on the expression profiles of MYC , TFAP4 , ATM and NLK also revealed a clear separation of the two groups. d . miRNA seq. Expression counts of hsa-miR-26a-5p, hsa-miR-27b-3p, hsa-miR-30b-5p, miR-17~92-cluster members (hsa-miR-19b-3p, and hsa-miR-92a-3p), and let-7-family miRs (hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7d-5p, hsa-let-7e-5p, and hsa-let-7 g-5p) in eBL tumor cells and GC B cells

Techniques Used: Expressing, Small Interfering RNA, Translocation Assay, Activity Assay, Activation Assay, RNA Sequencing Assay

25) Product Images from "Proliferation Cycle Causes Age Dependent Mitochondrial Deficiencies and Contributes to the Aging of Stem Cells"

Article Title: Proliferation Cycle Causes Age Dependent Mitochondrial Deficiencies and Contributes to the Aging of Stem Cells

Journal: Genes

doi: 10.3390/genes8120397

Mitochondrial DNA (mtDNA) rearrangements and impaired mtDNA replication in ovaries of aged flies. ( A ) Schematic drawing of Drosophila melanogaster (Dm.) mtDNA. Enzyme sites of HindIII (H) and XhoI (X) are indicated. Sizes of digested fragments are also labelled. ( B ) Representative gel image of rolling cycle amplification of mtDNA (arrowhead) and DNA marker (M, kb). ( C ) Pattern of rolling cycle amplification (RCA) amplified mtDNA digested by XhoI and HindIII. The 5.8 kb fragment spanning the AT-rich region was recovered for restriction fragment length polymorphism (RFLP) analysis. ( D ) Schematic map of SspI site on 5.8 kb AT-rich region. ( E ) densitometry plot of SspI digestion pattern of 5.8 fragments spanning the AT-rich region from young (2-day-old) and old (60-day-old) ovaries, analyzed by Agilent Bioanalyzer 2100. Note the difference of bands (open arrowheads) demonstrating the rearrangements of AT-rich regions in aged ovaries. DNA dye standard (closed arrowheads) and DNA ladder are marked (M, bp). ( F ) Representative images showing 5-ethynyl-2’-deoxyuridine (EdU) incorporation into mtDNA (green puncta, arrowheads) in ovaries (dashed line, anterior toward left) from young (2-day-old) and old (40-day-old) female flies. Note that the number of EdU puncta was dramatically reduced in germarium from old fly. Arrowhead: mtDNA; arrow: nuclear DNA; scale bar: 10 µm.
Figure Legend Snippet: Mitochondrial DNA (mtDNA) rearrangements and impaired mtDNA replication in ovaries of aged flies. ( A ) Schematic drawing of Drosophila melanogaster (Dm.) mtDNA. Enzyme sites of HindIII (H) and XhoI (X) are indicated. Sizes of digested fragments are also labelled. ( B ) Representative gel image of rolling cycle amplification of mtDNA (arrowhead) and DNA marker (M, kb). ( C ) Pattern of rolling cycle amplification (RCA) amplified mtDNA digested by XhoI and HindIII. The 5.8 kb fragment spanning the AT-rich region was recovered for restriction fragment length polymorphism (RFLP) analysis. ( D ) Schematic map of SspI site on 5.8 kb AT-rich region. ( E ) densitometry plot of SspI digestion pattern of 5.8 fragments spanning the AT-rich region from young (2-day-old) and old (60-day-old) ovaries, analyzed by Agilent Bioanalyzer 2100. Note the difference of bands (open arrowheads) demonstrating the rearrangements of AT-rich regions in aged ovaries. DNA dye standard (closed arrowheads) and DNA ladder are marked (M, bp). ( F ) Representative images showing 5-ethynyl-2’-deoxyuridine (EdU) incorporation into mtDNA (green puncta, arrowheads) in ovaries (dashed line, anterior toward left) from young (2-day-old) and old (40-day-old) female flies. Note that the number of EdU puncta was dramatically reduced in germarium from old fly. Arrowhead: mtDNA; arrow: nuclear DNA; scale bar: 10 µm.

Techniques Used: Amplification, Marker

26) Product Images from "Nucleotide-resolution DNA double-strand breaks mapping by next-generation sequencing"

Article Title: Nucleotide-resolution DNA double-strand breaks mapping by next-generation sequencing

Journal: Nature methods

doi: 10.1038/nmeth.2408

ASRs validation. ( a ) Fraction of input DNA captured by ChIP in regions with significant (grey highlight) vs. non-significant aphidicolin effect in HeLa cells treated (orange) or not (green) with aphidicolin. Mean ± s.d. are shown for n = 3 biological replicates. Genomic coordinates of amplicons analyzed by qPCR are reported. Chr: chromosome. Coord: genomic coordinate. ( b ) Comparison of aphidicolin effect measured by BLESS vs. ChIP in regions described in ( a ). Captured DNA ratio: ratio of captured DNA in aphidicolin-treated (A) vs. control (C) HeLa. R: Pearson’s correlation coefficient.
Figure Legend Snippet: ASRs validation. ( a ) Fraction of input DNA captured by ChIP in regions with significant (grey highlight) vs. non-significant aphidicolin effect in HeLa cells treated (orange) or not (green) with aphidicolin. Mean ± s.d. are shown for n = 3 biological replicates. Genomic coordinates of amplicons analyzed by qPCR are reported. Chr: chromosome. Coord: genomic coordinate. ( b ) Comparison of aphidicolin effect measured by BLESS vs. ChIP in regions described in ( a ). Captured DNA ratio: ratio of captured DNA in aphidicolin-treated (A) vs. control (C) HeLa. R: Pearson’s correlation coefficient.

Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

27) Product Images from "Large-Scale Low-Cost NGS Library Preparation Using a Robust Tn5 Purification and Tagmentation Protocol"

Article Title: Large-Scale Low-Cost NGS Library Preparation Using a Robust Tn5 Purification and Tagmentation Protocol

Journal: G3: Genes|Genomes|Genetics

doi: 10.1534/g3.117.300257

NGS-library preparation using the homemade Tn5 constructs. (A) Workflow of Tn5 loading, cDNA tagmentation, and subsequent NGS library preparation for duplex index (Illumina i5/i7) full-length cDNA sequencing. Tn5 molecules are shown as gray hexamers. The double-stranded part of the linker oligonucleotide, the mosaic element, is shown in gray with a yellow circle depicting the phosphorylated 3′ end. The 5′ overhangs as templates for the i5 or i7 index adapter primers are shown in red or blue, respectively. cDNA is shown as two parallel black lines with a 3′ poly(A) tail. The synthesis of the 5′ overhang complementary strand (gap-filling step during PCR amplification) is depicted as a dotted arrow. i5 index adapter primer is shown in dark blue, while i7 index adapter primer is shown in orange. Fragments that are lost during library preparation are transparent. (B) Bioanalyzer traces of NGS libraries processed with different concentrations of the in-house-produced Tn5 R27S,E54K,L372P using only homemade or inexpensive commercially available reagents for tagmentation and subsequent PCR reaction. Following the tagmentation protocol presented here, fragmentation of the cDNA works best when using Tn5 R27S,E54K,L372P at a concentration of 30 ng/μl. (C) Heat scatter plot showing the correlation of read counts between libraries processed with either in-house-produced Tn5 E54K,L372P or Tn5 R27S,E54K,L372P . Data of three technical replicates per condition were pooled for this analysis. (D) Heat scatter plot showing the correlation of gene counts between libraries processed using either in-house-produced Tn5 R27S,E54K,L372P and the protocol presented here or the Nextera XT DNA library preparation kit following the manufacturer’s instructions. Data of three technical replicates per condition were pooled for this analysis. (E) Pairwise correlation of read counts between three technical replicates (samples processed from the same cDNA on the same day) when using homemade Tn5 R27S,E54K,L372P and the tagmentation protocol presented here. The Pearson correlation of r = 0.99 between all samples demonstrates high reproducibility of both the enzyme and the protocol. (F) Heat map analysis of gene counts in technical replicates processed from the same cDNA but on different days. The color code indicates the Pearson correlation between samples (see legend on the right side). NGS, next-generation sequencing; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate.
Figure Legend Snippet: NGS-library preparation using the homemade Tn5 constructs. (A) Workflow of Tn5 loading, cDNA tagmentation, and subsequent NGS library preparation for duplex index (Illumina i5/i7) full-length cDNA sequencing. Tn5 molecules are shown as gray hexamers. The double-stranded part of the linker oligonucleotide, the mosaic element, is shown in gray with a yellow circle depicting the phosphorylated 3′ end. The 5′ overhangs as templates for the i5 or i7 index adapter primers are shown in red or blue, respectively. cDNA is shown as two parallel black lines with a 3′ poly(A) tail. The synthesis of the 5′ overhang complementary strand (gap-filling step during PCR amplification) is depicted as a dotted arrow. i5 index adapter primer is shown in dark blue, while i7 index adapter primer is shown in orange. Fragments that are lost during library preparation are transparent. (B) Bioanalyzer traces of NGS libraries processed with different concentrations of the in-house-produced Tn5 R27S,E54K,L372P using only homemade or inexpensive commercially available reagents for tagmentation and subsequent PCR reaction. Following the tagmentation protocol presented here, fragmentation of the cDNA works best when using Tn5 R27S,E54K,L372P at a concentration of 30 ng/μl. (C) Heat scatter plot showing the correlation of read counts between libraries processed with either in-house-produced Tn5 E54K,L372P or Tn5 R27S,E54K,L372P . Data of three technical replicates per condition were pooled for this analysis. (D) Heat scatter plot showing the correlation of gene counts between libraries processed using either in-house-produced Tn5 R27S,E54K,L372P and the protocol presented here or the Nextera XT DNA library preparation kit following the manufacturer’s instructions. Data of three technical replicates per condition were pooled for this analysis. (E) Pairwise correlation of read counts between three technical replicates (samples processed from the same cDNA on the same day) when using homemade Tn5 R27S,E54K,L372P and the tagmentation protocol presented here. The Pearson correlation of r = 0.99 between all samples demonstrates high reproducibility of both the enzyme and the protocol. (F) Heat map analysis of gene counts in technical replicates processed from the same cDNA but on different days. The color code indicates the Pearson correlation between samples (see legend on the right side). NGS, next-generation sequencing; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate.

Techniques Used: Next-Generation Sequencing, Construct, Sequencing, Polymerase Chain Reaction, Amplification, Produced, Concentration Assay

28) Product Images from "Targeted next generation sequencing for molecular diagnosis of Usher syndrome"

Article Title: Targeted next generation sequencing for molecular diagnosis of Usher syndrome

Journal: Orphanet Journal of Rare Diseases

doi: 10.1186/s13023-014-0168-7

Images obtained in the validation and quantification of the enriched target DNA with the 2100 Bioanalyzer system. A) Typical image obtained in most patients. B) Atypical image obtained in the patient RP-531, in whom CNV analysis could not be performed.
Figure Legend Snippet: Images obtained in the validation and quantification of the enriched target DNA with the 2100 Bioanalyzer system. A) Typical image obtained in most patients. B) Atypical image obtained in the patient RP-531, in whom CNV analysis could not be performed.

Techniques Used:

29) Product Images from "Exosomes maintain cellular homeostasis by excreting harmful DNA from cells"

Article Title: Exosomes maintain cellular homeostasis by excreting harmful DNA from cells

Journal: Nature Communications

doi: 10.1038/ncomms15287

Exosomes contain chromosome fragments. ( a ) transmission electron microscopy micrograph of MVE in pre-senescent TIG-3 cells following immuno-gold labelling for dsDNA. Gold particles are depicted as black dots. Right image shows a digitally zoomed area of exosome. ( b , c ) Exosomal DNA isolated from pre-senescent TIG-3 cells were subjected to size distribution analysis using Electrophoresis Bioanalyzer system ( b ) or to deep sequencing analysis ( c ). Genomic DNA of TIG-3 cells are also subjected to deep sequencing analysis, as control ( c ). The read count of each 500-kb bin was normalized to RPKM and corrected by the mappability ( c ). ( d ) Purified exosomes from pre-senescent TIG-3 cells were subjected to sucrose density-gradient separation followed by western blotting using antibodies shown right, NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles and quantitative PCR analysis for detection of genomic DNA fragments (GRM7 and GPC6).
Figure Legend Snippet: Exosomes contain chromosome fragments. ( a ) transmission electron microscopy micrograph of MVE in pre-senescent TIG-3 cells following immuno-gold labelling for dsDNA. Gold particles are depicted as black dots. Right image shows a digitally zoomed area of exosome. ( b , c ) Exosomal DNA isolated from pre-senescent TIG-3 cells were subjected to size distribution analysis using Electrophoresis Bioanalyzer system ( b ) or to deep sequencing analysis ( c ). Genomic DNA of TIG-3 cells are also subjected to deep sequencing analysis, as control ( c ). The read count of each 500-kb bin was normalized to RPKM and corrected by the mappability ( c ). ( d ) Purified exosomes from pre-senescent TIG-3 cells were subjected to sucrose density-gradient separation followed by western blotting using antibodies shown right, NanoSight analysis (NTA) for quantitative measurement of isolated exosome particles and quantitative PCR analysis for detection of genomic DNA fragments (GRM7 and GPC6).

Techniques Used: Transmission Assay, Electron Microscopy, Isolation, Electrophoresis, Sequencing, Purification, Western Blot, Real-time Polymerase Chain Reaction

30) Product Images from "Circulating Tumor DNA Mutation Profiling by Targeted Next Generation Sequencing Provides Guidance for Personalized Treatments in Multiple Cancer Types"

Article Title: Circulating Tumor DNA Mutation Profiling by Targeted Next Generation Sequencing Provides Guidance for Personalized Treatments in Multiple Cancer Types

Journal: Scientific Reports

doi: 10.1038/s41598-017-00520-1

Workflow of targeted NGS-based mutation profiling. ( a ) Genomic DNA is extracted from multiple sample types. ( b ) Whole-genome libraries are prepared from fragmented genomic DNA or cfDNA, followed by hybridization capture with biotinylated DNA probes to establish target-enriched sequencing libraries for NGS. ( c ) Sequencing data undergoes quality control (QC), mapping and bioinformatic analysis to identify different classes of genomic aberrations. ( d ) Mutations identified are filtered and annotated according to related databases, and their clinical significances are interpreted in the final report.
Figure Legend Snippet: Workflow of targeted NGS-based mutation profiling. ( a ) Genomic DNA is extracted from multiple sample types. ( b ) Whole-genome libraries are prepared from fragmented genomic DNA or cfDNA, followed by hybridization capture with biotinylated DNA probes to establish target-enriched sequencing libraries for NGS. ( c ) Sequencing data undergoes quality control (QC), mapping and bioinformatic analysis to identify different classes of genomic aberrations. ( d ) Mutations identified are filtered and annotated according to related databases, and their clinical significances are interpreted in the final report.

Techniques Used: Next-Generation Sequencing, Mutagenesis, Hybridization, Sequencing

31) Product Images from "Expanded subgenomic mRNA transcriptome and coding capacity of a nidovirus"

Article Title: Expanded subgenomic mRNA transcriptome and coding capacity of a nidovirus

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1706696114

Amplification and cloning of sg mRNA leader–body junctions generated from additional functional TRSs located in the genomic region between identified TRS5 and TRS6. ( A ) Diagram indicating the positions of the primers used and the estimated size for the amplified leader–body junction sequences (thick black line). The white open box represents ORF5, and the short black box represents the leader sequence in transcribed sg mRNAs. ( B ) MA104 cells were either mock-infected (M) or infected with SHFVic at an MOI of 1. At 24 hpi, total intracellular RNA was extracted and subjected to RT-PCR, and the products were separated on a 2% DNA gel. The band with the size estimated for the leader–body junction in sg mRNA5 produced from the known TRS5 is indicated by an arrow. PCR bands with sizes estimated for the leader–body junctions of ∼1.7-kb sg mRNAs are indicated by a bracket. L, ladder. ( C ) The bracketed region of the gel was excised, and the DNA was extracted and cloned into a TA vector. Forty colonies were randomly selected and subjected to restriction digestion, and the inserts were separated by gel electrophoresis. The results from 10 representative clones are shown. L, ladder. ( D ) Diagram showing the locations of the known and previously unreported body TRSs. The TRSs are indicated by black vertical bars. The previously unreported functional TRSs are within a dotted line box. The ORFs encoded by the individual sg mRNAs are indicated by white open boxes. 5-C, ORF5-C-68aa; L, leader region.
Figure Legend Snippet: Amplification and cloning of sg mRNA leader–body junctions generated from additional functional TRSs located in the genomic region between identified TRS5 and TRS6. ( A ) Diagram indicating the positions of the primers used and the estimated size for the amplified leader–body junction sequences (thick black line). The white open box represents ORF5, and the short black box represents the leader sequence in transcribed sg mRNAs. ( B ) MA104 cells were either mock-infected (M) or infected with SHFVic at an MOI of 1. At 24 hpi, total intracellular RNA was extracted and subjected to RT-PCR, and the products were separated on a 2% DNA gel. The band with the size estimated for the leader–body junction in sg mRNA5 produced from the known TRS5 is indicated by an arrow. PCR bands with sizes estimated for the leader–body junctions of ∼1.7-kb sg mRNAs are indicated by a bracket. L, ladder. ( C ) The bracketed region of the gel was excised, and the DNA was extracted and cloned into a TA vector. Forty colonies were randomly selected and subjected to restriction digestion, and the inserts were separated by gel electrophoresis. The results from 10 representative clones are shown. L, ladder. ( D ) Diagram showing the locations of the known and previously unreported body TRSs. The TRSs are indicated by black vertical bars. The previously unreported functional TRSs are within a dotted line box. The ORFs encoded by the individual sg mRNAs are indicated by white open boxes. 5-C, ORF5-C-68aa; L, leader region.

Techniques Used: Amplification, Clone Assay, Generated, Functional Assay, Sequencing, Infection, Reverse Transcription Polymerase Chain Reaction, Produced, Polymerase Chain Reaction, Plasmid Preparation, Nucleic Acid Electrophoresis

32) Product Images from "Improved Protocols for Illumina Sequencing"

Article Title: Improved Protocols for Illumina Sequencing

Journal: Current protocols in human genetics / editorial board, Jonathan L. Haines ... [et al.]

doi: 10.1002/0471142905.hg1802s62

Effect of AMPure XP ratios on fragment size selection. 1 μg of DNA was sheared to giver fragments from 20 to 400bp (average 160bp). Next the DNA was incubated with different AMPure ratios. The size distribution of fragments after AMPure clean up were analysed by electrophoresis using an Agilent Bioanalyzer High Sensitivity DNA chip. (A) AMPure bead to DNA ratios varying from 2.5× beads to DNA up to 1.0× beads to DNA (B) AMPure bead to DNA ratios varying from 1.5× beads to DNA up to 0.6× beads to DNA.
Figure Legend Snippet: Effect of AMPure XP ratios on fragment size selection. 1 μg of DNA was sheared to giver fragments from 20 to 400bp (average 160bp). Next the DNA was incubated with different AMPure ratios. The size distribution of fragments after AMPure clean up were analysed by electrophoresis using an Agilent Bioanalyzer High Sensitivity DNA chip. (A) AMPure bead to DNA ratios varying from 2.5× beads to DNA up to 1.0× beads to DNA (B) AMPure bead to DNA ratios varying from 1.5× beads to DNA up to 0.6× beads to DNA.

Techniques Used: Selection, Incubation, Electrophoresis, Chromatin Immunoprecipitation

33) Product Images from "gbpA as a Novel qPCR Target for the Species-Specific Detection of Vibrio cholerae O1, O139, Non-O1/Non-O139 in Environmental, Stool, and Historical Continuous Plankton Recorder Samples"

Article Title: gbpA as a Novel qPCR Target for the Species-Specific Detection of Vibrio cholerae O1, O139, Non-O1/Non-O139 in Environmental, Stool, and Historical Continuous Plankton Recorder Samples

Journal: PLoS ONE

doi: 10.1371/journal.pone.0123983

Performance of the qPCR assay for detection of V . cholerae in artificially degraded DNA samples. (A) Electropherogram plot obtained by Agilent Bioanalyzer analysis of artificially fragmented genomic DNA of V . cholerae ATCC 39315. (B) Plot of mean Cq-values from three replicates tested against the V . cholerae artificially fragmented DNA inputs. Error bars indicate the standard deviations of the means.
Figure Legend Snippet: Performance of the qPCR assay for detection of V . cholerae in artificially degraded DNA samples. (A) Electropherogram plot obtained by Agilent Bioanalyzer analysis of artificially fragmented genomic DNA of V . cholerae ATCC 39315. (B) Plot of mean Cq-values from three replicates tested against the V . cholerae artificially fragmented DNA inputs. Error bars indicate the standard deviations of the means.

Techniques Used: Real-time Polymerase Chain Reaction

Related Articles

Centrifugation:

Article Title: A quantitative analysis of the potential biomarkers of non-small cell lung cancer by circulating cell-free DNA
Article Snippet: The process consisted of several steps as follows: i) capturing cfDNA in the polymer by mixing serum/plasma with 30 µl of VCR-1 and 150 µl of VCR-2, followed by centrifugation at 18,500 × g for 3 min at room temperature; ii) washing the polymer/DNA complex with 1 ml of ddH2 O; iii) adding 400 µl of lysis solution SE to dissolve the pellet; iv) adding 50 µl of PK and incubating the samples for 15 min at 70°C; v) adding 400 µl of binding solution SBS and transferring the samples to spin filters; vi) washing the samples by adding 500 µl of washing solution GS and 650 µl of washing solution BS, followed by centrifugation at 13,500 × g for 1 min at room temperature; and vii) eluting cfDNA in 50 µl of water and storing the cfDNA at −80°C until further analysis. .. 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.

Amplification:

Article Title: Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads
Article Snippet: .. We checked the length distribution of amplified cDNA with an Agilent High Sensitivity DNA Kit (Agilent). .. The typical average size of the amplified cDNA in Quartz-Seq2 was approximately 1400 bp (Additional file : Figure S2c).

Article Title: Genome-wide co-localization of active EGFR and downstream ERK pathway kinases mirrors mitogen-inducible RNA polymerase 2 genomic occupancy
Article Snippet: Adenylated fragments were ligated with Illumina's indexed adapters and then amplified during 18-cycles PCR for selective enrichment. .. 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.

Article Title: Ecophylogeny of the endospheric root fungal microbiome of co-occurring Agrostis stolonifera
Article Snippet: Paragraph title: Nucleic acid extraction and 18S rRNA amplicon sequencing ... RNA and DNA quality was checked using the RNA 6000 Pico kit or the High sensitivity DNA kit, respectively, on a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA).

Article Title: Investigation of Gene Expressions of Myeloma Cells in the Bone Marrow of Multiple Myeloma Patients by Transcriptome Analysis
Article Snippet: The quality of the libraries was validated using the Agilent High Sensitivity DNA kit on the Agilent 2100 Bioanalyzer ( ). .. Each library was amplified by Emulsion polymerase chain reaction of ion sphere particles (ISPs).

Blocking Assay:

Article Title: Generating Exome Enriched Sequencing Libraries from Formalin-Fixed, Paraffin-Embedded Tissue DNA for Next Generation Sequencing
Article Snippet: .. Universal Blocker Oligonucleotide (Integrated DNA Technology) – See Reagents and Solutions Indexed Blocker Oligonucleotide (Integrated DNA Technology) – See Reagents and Solutions Nuclease Free Water (ThermoFisher Scientific; pn: AM9930) SureSelect XT Human All Exon Kit (Agilent; pn:5190-8863) SureSelect Hyb #1, 2, 3, 4 SureSelect Block # 1, 2 RNase Block SureSelect Human All Exon Capture Sure Select Binding Buffer Sure Select Wash Buffer I Sure Select Wash Buffer II Dynabeads My One Streptavidin T1 (ThermoFisher Scientific; pn: 65602) HiFi HotStart ReadyMix (2×) (KapaBiosystems; pn: KK2612) PCR Primer 1 (Integrated DNA Technology) – See Reagents and Solutions PCR Primer 2 (Integrated DNA Technology) – See Reagents and Solutions Agencourt Ampure XP (Beckman Coulter; pn: ) Ethanol 100% molecular biology grade Elution Buffer (Qiagen; pn: 19086) 2100 BioAnalyzer (Agilent) Vacuum concentrator MicroAmp Optical 8-Cap Strips (ThermoFisher Scientific; pn: 4323032) High Sensitivity DNA Kit (Agilent; 5067-4626) MicroAmp Optical 96 well Reaction Plate (ThermoFisher Scientific; pn: N8010560) MicroAmp Clear Adhesive Film (ThermoFisher Scientific; pn: 4306311) DynaMag-2 Magnet (Invitrogen; pn: 12321D) DynaMag-96 Side Skirted Plate Magnet (Invitrogen; pn: 12027 or 12331D) DNA LoBind Tubes (Eppendorf; pn: 22431021) Tube Rocker BD Clay Adams Nutator Mixer (BD Diagnostics; pn: 421105) Torrey Pines Echotherm Heat Block (Torrey Pines) ..

Real-time Polymerase Chain Reaction:

Article Title: Genome-wide co-localization of active EGFR and downstream ERK pathway kinases mirrors mitogen-inducible RNA polymerase 2 genomic occupancy
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. .. Samples were sequenced during single 50 bp read run on Illumina HiSeq 2500 system in High Output mode (using TruSeq SR Cluster Kit v3-cBot-HS and TruSeq SBS Kit v3-HS) and Rapid mode (using TruSeq Rapid Duo Sample Loading Kit, TruSeq Rapid SR Cluster Kit and TruSeq Rapid SBS Kit).

In Silico:

Article Title: Ecophylogeny of the endospheric root fungal microbiome of co-occurring Agrostis stolonifera
Article Snippet: RNA and DNA quality was checked using the RNA 6000 Pico kit or the High sensitivity DNA kit, respectively, on a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). .. These particular primers were chosen among a variety of candidate primers after an in silico analysis, using Primer Search ( ).

Modification:

Article Title: Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads
Article Snippet: We checked the length distribution of amplified cDNA with an Agilent High Sensitivity DNA Kit (Agilent). .. In the case of the usage of the v3.2 RT primer in Quartz-Seq2, we modified the above steps as follows.

Article Title: Ecophylogeny of the endospheric root fungal microbiome of co-occurring Agrostis stolonifera
Article Snippet: RNA and DNA were co-extracted from the nineteen samples using a modified RNeasy Plant Mini kit (Qiagen, Hilden, Germany) protocol. .. RNA and DNA quality was checked using the RNA 6000 Pico kit or the High sensitivity DNA kit, respectively, on a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA).

Ligation:

Article Title: Generating Exome Enriched Sequencing Libraries from Formalin-Fixed, Paraffin-Embedded Tissue DNA for Next Generation Sequencing
Article Snippet: .. Hyper Prep Kit (KapaBiosystems; pn: KK8504) End Repair/A Tailing Buffer End Repair/A Tailing Enzyme Ligation Buffer Kapa T4 DNA Ligase Kapa HiFi Master Mix Kapa Primer Premix Indexed Adapter oligonucleotide (Integrated DNA Technology) – See Reagents and Solutions 20% PEG/2.5M NaCl solution – See Reagents and Solutions Ethanol 100% molecular biology grade Nuclease Free Water (ThermoFisher Scientific; pn: AM9930) Agencourt Ampure XP (Beckman Coulter; pn: ) MicroAmp Optical 96 well Reaction Plate (ThermoFisher Scientific; pn: N8010560) MicroAmp Clear Adhesive Film (ThermoFisher Scientific; pn: 4306311) 2100 BioAnalyzer (Agilent) Elution Buffer (Qiagen; pn: 19086) High Sensitivity DNA Kit (Agilent; 5067-4626) Vortex Centrifuge Thermal cycler DynaMag-96 Side Skirted Plate Magnet (Invitrogen; pn:12027 or 12331D) ..

Transferring:

Article Title: A quantitative analysis of the potential biomarkers of non-small cell lung cancer by circulating cell-free DNA
Article Snippet: The process consisted of several steps as follows: i) capturing cfDNA in the polymer by mixing serum/plasma with 30 µl of VCR-1 and 150 µl of VCR-2, followed by centrifugation at 18,500 × g for 3 min at room temperature; ii) washing the polymer/DNA complex with 1 ml of ddH2 O; iii) adding 400 µl of lysis solution SE to dissolve the pellet; iv) adding 50 µl of PK and incubating the samples for 15 min at 70°C; v) adding 400 µl of binding solution SBS and transferring the samples to spin filters; vi) washing the samples by adding 500 µl of washing solution GS and 650 µl of washing solution BS, followed by centrifugation at 13,500 × g for 1 min at room temperature; and vii) eluting cfDNA in 50 µl of water and storing the cfDNA at −80°C until further analysis. .. 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.

Sequencing:

Article Title: Bead-linked transposomes enable a normalization-free workflow for NGS library preparation
Article Snippet: For DNA inputs of 100 to 500 ng, libraries were pooled by volume (5 μl per sample; up to 96 samples) into a 1.5 mL tube prior to sequencing. .. 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).

Article Title: Genome-wide co-localization of active EGFR and downstream ERK pathway kinases mirrors mitogen-inducible RNA polymerase 2 genomic occupancy
Article Snippet: Sequencing DNA libraries were prepared from up to 20 ng of DNA using TruSeq™ ChIP Sample Prep Kit (Illumina Inc.), according to a standard protocol with slight modifications. .. 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.

Article Title: Ecophylogeny of the endospheric root fungal microbiome of co-occurring Agrostis stolonifera
Article Snippet: Paragraph title: Nucleic acid extraction and 18S rRNA amplicon sequencing ... RNA and DNA quality was checked using the RNA 6000 Pico kit or the High sensitivity DNA kit, respectively, on a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA).

Binding Assay:

Article Title: Generating Exome Enriched Sequencing Libraries from Formalin-Fixed, Paraffin-Embedded Tissue DNA for Next Generation Sequencing
Article Snippet: .. Universal Blocker Oligonucleotide (Integrated DNA Technology) – See Reagents and Solutions Indexed Blocker Oligonucleotide (Integrated DNA Technology) – See Reagents and Solutions Nuclease Free Water (ThermoFisher Scientific; pn: AM9930) SureSelect XT Human All Exon Kit (Agilent; pn:5190-8863) SureSelect Hyb #1, 2, 3, 4 SureSelect Block # 1, 2 RNase Block SureSelect Human All Exon Capture Sure Select Binding Buffer Sure Select Wash Buffer I Sure Select Wash Buffer II Dynabeads My One Streptavidin T1 (ThermoFisher Scientific; pn: 65602) HiFi HotStart ReadyMix (2×) (KapaBiosystems; pn: KK2612) PCR Primer 1 (Integrated DNA Technology) – See Reagents and Solutions PCR Primer 2 (Integrated DNA Technology) – See Reagents and Solutions Agencourt Ampure XP (Beckman Coulter; pn: ) Ethanol 100% molecular biology grade Elution Buffer (Qiagen; pn: 19086) 2100 BioAnalyzer (Agilent) Vacuum concentrator MicroAmp Optical 8-Cap Strips (ThermoFisher Scientific; pn: 4323032) High Sensitivity DNA Kit (Agilent; 5067-4626) MicroAmp Optical 96 well Reaction Plate (ThermoFisher Scientific; pn: N8010560) MicroAmp Clear Adhesive Film (ThermoFisher Scientific; pn: 4306311) DynaMag-2 Magnet (Invitrogen; pn: 12321D) DynaMag-96 Side Skirted Plate Magnet (Invitrogen; pn: 12027 or 12331D) DNA LoBind Tubes (Eppendorf; pn: 22431021) Tube Rocker BD Clay Adams Nutator Mixer (BD Diagnostics; pn: 421105) Torrey Pines Echotherm Heat Block (Torrey Pines) ..

Article Title: A quantitative analysis of the potential biomarkers of non-small cell lung cancer by circulating cell-free DNA
Article Snippet: The process consisted of several steps as follows: i) capturing cfDNA in the polymer by mixing serum/plasma with 30 µl of VCR-1 and 150 µl of VCR-2, followed by centrifugation at 18,500 × g for 3 min at room temperature; ii) washing the polymer/DNA complex with 1 ml of ddH2 O; iii) adding 400 µl of lysis solution SE to dissolve the pellet; iv) adding 50 µl of PK and incubating the samples for 15 min at 70°C; v) adding 400 µl of binding solution SBS and transferring the samples to spin filters; vi) washing the samples by adding 500 µl of washing solution GS and 650 µl of washing solution BS, followed by centrifugation at 13,500 × g for 1 min at room temperature; and vii) eluting cfDNA in 50 µl of water and storing the cfDNA at −80°C until further analysis. .. 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.

Molecular Weight:

Article Title: New evidence that a large proportion of human blood plasma cell-free DNA is localized in exosomes
Article Snippet: RNase treatment had no visible effect on low intensity low molecular weight band indicating that band is DNA present in exosomes. .. RNase treated or not treated exosome DNA was also analyzed by Agilent Bioanalyzer using Agilent High Sensitivity DNA kit. shows results from two donors.

DNA Extraction:

Article Title: New evidence that a large proportion of human blood plasma cell-free DNA is localized in exosomes
Article Snippet: It is clear that RNase treatment removed a significant amount of high intensity high molecular weight band indicating that band is RNA co-purified during DNA extraction. .. RNase treated or not treated exosome DNA was also analyzed by Agilent Bioanalyzer using Agilent High Sensitivity DNA kit. shows results from two donors.

Article Title: A quantitative analysis of the potential biomarkers of non-small cell lung cancer by circulating cell-free DNA
Article Snippet: cfDNA extraction and fragment analysis Circulating DNA was separated from 1 ml serum and plasma samples with the PME free-circulating DNA extraction kit (Analytik Jena AG, Jena, Germany), according to the manufacturer's instructions for up to 1-ml extractions with the lysis solution SE/binding solution SBS mechanism. .. 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.

RNA Sequencing Assay:

Article Title: Investigation of Gene Expressions of Myeloma Cells in the Bone Marrow of Multiple Myeloma Patients by Transcriptome Analysis
Article Snippet: Paragraph title: Library preparation and RNA sequencing ... The quality of the libraries was validated using the Agilent High Sensitivity DNA kit on the Agilent 2100 Bioanalyzer ( ).

Fluorescence:

Article Title: New evidence that a large proportion of human blood plasma cell-free DNA is localized in exosomes
Article Snippet: Quantification of DNA and RNA extracted from exosome pellet using fluorescence based assays showed that RNA concentration in exosomes are 20–30-fold higher compared to exosome DNA concentration (data not shown). .. RNase treated or not treated exosome DNA was also analyzed by Agilent Bioanalyzer using Agilent High Sensitivity DNA kit. shows results from two donors.

Isolation:

Article Title: Quantification of circulating cell-free DNA to predict patient survival in non-small-cell lung cancer
Article Snippet: Subsequently, the supernatants were stored at −80°C until use. cfDNA was isolated from serum aliquots (500 μL) by means of a QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany) with a QIAvac 24 Plus vacuum manifold, in accordance with the manufacturer’s instructions. .. Subsequently, the circulating cfDNA concentration was quantified with a Qubit 2.0 Fluorometer and the Agilent High Sensitivity DNA kit (Agilent Technologies).

Purification:

Article Title: Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads
Article Snippet: Finally, we obtained 32 μL of purified cDNA. .. We checked the length distribution of amplified cDNA with an Agilent High Sensitivity DNA Kit (Agilent).

Article Title: Quantification of circulating cell-free DNA to predict patient survival in non-small-cell lung cancer
Article Snippet: Paragraph title: Circulating cfDNA purification and quantification ... Subsequently, the circulating cfDNA concentration was quantified with a Qubit 2.0 Fluorometer and the Agilent High Sensitivity DNA kit (Agilent Technologies).

Polymerase Chain Reaction:

Article Title: Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads
Article Snippet: We added 32 μL of 3 M sodium acetate (pH 5.2) and 6420 μL PB-Buffer (Qiagen) to the PCR solution. .. We checked the length distribution of amplified cDNA with an Agilent High Sensitivity DNA Kit (Agilent).

Article Title: Genome-wide co-localization of active EGFR and downstream ERK pathway kinases mirrors mitogen-inducible RNA polymerase 2 genomic occupancy
Article Snippet: Adenylated fragments were ligated with Illumina's indexed adapters and then amplified during 18-cycles PCR for selective enrichment. .. 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.

Article Title: Ecophylogeny of the endospheric root fungal microbiome of co-occurring Agrostis stolonifera
Article Snippet: For RNA extraction half of the total nucleic extracts were treated with RQ1 DNAse (Promega, Madison, WI, USA) and the complete elimination of DNA was confirmed by 18S rRNA targeting PCR. .. RNA and DNA quality was checked using the RNA 6000 Pico kit or the High sensitivity DNA kit, respectively, on a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA).

Article Title: Generating Exome Enriched Sequencing Libraries from Formalin-Fixed, Paraffin-Embedded Tissue DNA for Next Generation Sequencing
Article Snippet: .. Universal Blocker Oligonucleotide (Integrated DNA Technology) – See Reagents and Solutions Indexed Blocker Oligonucleotide (Integrated DNA Technology) – See Reagents and Solutions Nuclease Free Water (ThermoFisher Scientific; pn: AM9930) SureSelect XT Human All Exon Kit (Agilent; pn:5190-8863) SureSelect Hyb #1, 2, 3, 4 SureSelect Block # 1, 2 RNase Block SureSelect Human All Exon Capture Sure Select Binding Buffer Sure Select Wash Buffer I Sure Select Wash Buffer II Dynabeads My One Streptavidin T1 (ThermoFisher Scientific; pn: 65602) HiFi HotStart ReadyMix (2×) (KapaBiosystems; pn: KK2612) PCR Primer 1 (Integrated DNA Technology) – See Reagents and Solutions PCR Primer 2 (Integrated DNA Technology) – See Reagents and Solutions Agencourt Ampure XP (Beckman Coulter; pn: ) Ethanol 100% molecular biology grade Elution Buffer (Qiagen; pn: 19086) 2100 BioAnalyzer (Agilent) Vacuum concentrator MicroAmp Optical 8-Cap Strips (ThermoFisher Scientific; pn: 4323032) High Sensitivity DNA Kit (Agilent; 5067-4626) MicroAmp Optical 96 well Reaction Plate (ThermoFisher Scientific; pn: N8010560) MicroAmp Clear Adhesive Film (ThermoFisher Scientific; pn: 4306311) DynaMag-2 Magnet (Invitrogen; pn: 12321D) DynaMag-96 Side Skirted Plate Magnet (Invitrogen; pn: 12027 or 12331D) DNA LoBind Tubes (Eppendorf; pn: 22431021) Tube Rocker BD Clay Adams Nutator Mixer (BD Diagnostics; pn: 421105) Torrey Pines Echotherm Heat Block (Torrey Pines) ..

Article Title: Investigation of Gene Expressions of Myeloma Cells in the Bone Marrow of Multiple Myeloma Patients by Transcriptome Analysis
Article Snippet: The quality of the libraries was validated using the Agilent High Sensitivity DNA kit on the Agilent 2100 Bioanalyzer ( ). .. Each library was amplified by Emulsion polymerase chain reaction of ion sphere particles (ISPs).

Construct:

Article Title: Investigation of Gene Expressions of Myeloma Cells in the Bone Marrow of Multiple Myeloma Patients by Transcriptome Analysis
Article Snippet: The libraries were constructed using the Ion Total RNA-seq V2 kit protocol for transcriptome profiling of low-input RNA samples (April 2011). .. The quality of the libraries was validated using the Agilent High Sensitivity DNA kit on the Agilent 2100 Bioanalyzer ( ).

Sample Prep:

Article Title: Genome-wide co-localization of active EGFR and downstream ERK pathway kinases mirrors mitogen-inducible RNA polymerase 2 genomic occupancy
Article Snippet: Sequencing DNA libraries were prepared from up to 20 ng of DNA using TruSeq™ ChIP Sample Prep Kit (Illumina Inc.), according to a standard protocol with slight modifications. .. 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.

Chromatin Immunoprecipitation:

Article Title: Genome-wide co-localization of active EGFR and downstream ERK pathway kinases mirrors mitogen-inducible RNA polymerase 2 genomic occupancy
Article Snippet: Sequencing DNA libraries were prepared from up to 20 ng of DNA using TruSeq™ ChIP Sample Prep Kit (Illumina Inc.), according to a standard protocol with slight modifications. .. 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.

Software:

Article Title: Genome-wide co-localization of active EGFR and downstream ERK pathway kinases mirrors mitogen-inducible RNA polymerase 2 genomic occupancy
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. .. Base calling, demultiplexing and generation of fastq files was carried on using Illumina RTA and bcl2fastq software.

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
Article Snippet: 2.4 Measurement of global concentration and fragment size of cfDNA 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). .. Agilent 2100 Expert software (version B.02.08) offers a smear analysis with an integrator feature that allows precise measurement of the smear region.

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
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. .. The concentration and molarity of each specified region was normalized by a ladder and by lower and upper markers.

Electrophoresis:

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
Article Snippet: The size distribution of plasma DNA was examined by a capillary electrophoresis system. .. 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.

RNA Extraction:

Article Title: Ecophylogeny of the endospheric root fungal microbiome of co-occurring Agrostis stolonifera
Article Snippet: For RNA extraction half of the total nucleic extracts were treated with RQ1 DNAse (Promega, Madison, WI, USA) and the complete elimination of DNA was confirmed by 18S rRNA targeting PCR. .. RNA and DNA quality was checked using the RNA 6000 Pico kit or the High sensitivity DNA kit, respectively, on a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA).

Agarose Gel Electrophoresis:

Article Title: New evidence that a large proportion of human blood plasma cell-free DNA is localized in exosomes
Article Snippet: Paragraph title: Analysis of exosome DNA by agarose gel electrophoresis ... RNase treated or not treated exosome DNA was also analyzed by Agilent Bioanalyzer using Agilent High Sensitivity DNA kit. shows results from two donors.

Next-Generation Sequencing:

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

Concentration Assay:

Article Title: Quantification of circulating cell-free DNA to predict patient survival in non-small-cell lung cancer
Article Snippet: .. Subsequently, the circulating cfDNA concentration was quantified with a Qubit 2.0 Fluorometer and the Agilent High Sensitivity DNA kit (Agilent Technologies). ..

Article Title: New evidence that a large proportion of human blood plasma cell-free DNA is localized in exosomes
Article Snippet: Quantification of DNA and RNA extracted from exosome pellet using fluorescence based assays showed that RNA concentration in exosomes are 20–30-fold higher compared to exosome DNA concentration (data not shown). .. RNase treated or not treated exosome DNA was also analyzed by Agilent Bioanalyzer using Agilent High Sensitivity DNA kit. shows results from two donors.

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
Article Snippet: .. 2.4 Measurement of global concentration and fragment size of cfDNA 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). .. Agilent 2100 Expert software (version B.02.08) offers a smear analysis with an integrator feature that allows precise measurement of the smear region.

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
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. .. The concentration and molarity of each specified region was normalized by a ladder and by lower and upper markers.

Article Title: Adapting the Smart-seq2 Protocol for Robust Single Worm RNA-seq
Article Snippet: Paragraph title: E. Measurement of cDNA concentration and sample quality check ... Run BioAnalyzer (BA) on the cDNA following the protocol for the Agilent High Sensitivity DNA Kit.

MicroChIP Assay:

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
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. .. The concentration and molarity of each specified region was normalized by a ladder and by lower and upper markers.

ChIP-sequencing:

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

Lysis:

Article Title: A quantitative analysis of the potential biomarkers of non-small cell lung cancer by circulating cell-free DNA
Article Snippet: The process consisted of several steps as follows: i) capturing cfDNA in the polymer by mixing serum/plasma with 30 µl of VCR-1 and 150 µl of VCR-2, followed by centrifugation at 18,500 × g for 3 min at room temperature; ii) washing the polymer/DNA complex with 1 ml of ddH2 O; iii) adding 400 µl of lysis solution SE to dissolve the pellet; iv) adding 50 µl of PK and incubating the samples for 15 min at 70°C; v) adding 400 µl of binding solution SBS and transferring the samples to spin filters; vi) washing the samples by adding 500 µl of washing solution GS and 650 µl of washing solution BS, followed by centrifugation at 13,500 × g for 1 min at room temperature; and vii) eluting cfDNA in 50 µl of water and storing the cfDNA at −80°C until further analysis. .. 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.

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    Agilent technologies high sensitivity dna kit
    Changes in the <t>cfDNA</t> 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 <t>DNA;</t> 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.
    High Sensitivity Dna Kit, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 99/100, based on 359 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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