dna extracts  (Covaris)


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    Name:
    E220 Focused ultrasonicator
    Description:
    High performance Ultrasonicator for samples of mass 1000mg and or volume 10ml Automated batch sample preparation system serial process
    Catalog Number:
    500239
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    Size:
    24 W x 30 D x 19 H
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    Structured Review

    Covaris dna extracts
    E220 Focused ultrasonicator
    High performance Ultrasonicator for samples of mass 1000mg and or volume 10ml Automated batch sample preparation system serial process
    https://www.bioz.com/result/dna extracts/product/Covaris
    Average 93 stars, based on 207 article reviews
    Price from $9.99 to $1999.99
    dna extracts - by Bioz Stars, 2020-05
    93/100 stars

    Images

    1) Product Images from "Ligation Bias in Illumina Next-Generation DNA Libraries: Implications for Sequencing Ancient Genomes"

    Article Title: Ligation Bias in Illumina Next-Generation DNA Libraries: Implications for Sequencing Ancient Genomes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0078575

    A low adapter concentration magnifies the base composition bias for AT libraries. Aliquots of E. coli DNA extracts were sheared using the Covaris E210 sonicator, size selected, and built into AT libraries using a low (“low”, 0.012 µM) or a standard (“std”, 0.6 µM) adapter concentration. We report the base composition observed at the start (position +1) and the end (position N) of sequences for reads mapping with high quality a unique position of the E. coli NC_010473 genome. The base compositions of the first nucleotide located upstream (position –1) and downstream (position N+1) read alignments are also provided. The average base composition is reported with dashed lines and is calculated using base counts within the reference genome.
    Figure Legend Snippet: A low adapter concentration magnifies the base composition bias for AT libraries. Aliquots of E. coli DNA extracts were sheared using the Covaris E210 sonicator, size selected, and built into AT libraries using a low (“low”, 0.012 µM) or a standard (“std”, 0.6 µM) adapter concentration. We report the base composition observed at the start (position +1) and the end (position N) of sequences for reads mapping with high quality a unique position of the E. coli NC_010473 genome. The base compositions of the first nucleotide located upstream (position –1) and downstream (position N+1) read alignments are also provided. The average base composition is reported with dashed lines and is calculated using base counts within the reference genome.

    Techniques Used: Concentration Assay

    Base composition bias: AT versus BE libraries. Fresh aliquots of E. coli DNA extracts were sheared using the Covaris E210 sonicator, size selected, and built into AT and BE libraries (adapter concentration = 0.6 µM). We report the base composition observed at the first 10 (positions 1 to 10) and last 10 (positions N-9 to N) nucleotide positions within sequence reads mapping with high quality a unique position of the E. coli NC_010473 genome. The genomic composition of the 10 nucleotides located upstream (positions –10 to –1) and downstream (positions N+1 to N+10) DNA inserts are also provided.
    Figure Legend Snippet: Base composition bias: AT versus BE libraries. Fresh aliquots of E. coli DNA extracts were sheared using the Covaris E210 sonicator, size selected, and built into AT and BE libraries (adapter concentration = 0.6 µM). We report the base composition observed at the first 10 (positions 1 to 10) and last 10 (positions N-9 to N) nucleotide positions within sequence reads mapping with high quality a unique position of the E. coli NC_010473 genome. The genomic composition of the 10 nucleotides located upstream (positions –10 to –1) and downstream (positions N+1 to N+10) DNA inserts are also provided.

    Techniques Used: Concentration Assay, Sequencing

    2) Product Images from "Semiconductor-based sequencing of genome-wide DNA methylation states"

    Article Title: Semiconductor-based sequencing of genome-wide DNA methylation states

    Journal: Epigenetics

    doi: 10.1080/15592294.2014.1003747

    Scalability of Ion Torrent-compatible MeDIP-Seq protocol. (a) Global 5-methylcytosine (5mC) content measured as a percentage of the genome in DNA from WT and DKO cells determined by an ELISA assay. (b) Number of peaks identified by MACS v2.1.0 software 64 from MeDIP-Seq data in WT and DKO cells. (c) Sequence coverage of genome-wide CpGs for WT and DKO cells on the Ion Torrent Proton sequencer calculated using MEDIPS v1.14.0 software. 39 Pie charts illustrate the fraction of CpGs covered by the indicated reads according to their fold-coverage in relation to the total genomic CpG content from MeDIP-Seq libraries of the indicated sample sequenced on the Proton with the total number of non-redundant mapped reads in parentheses. (d) Sequence coverage of genome-wide CpGs for WT and DKO cells in MeDIP-Seq on the Ion Torrent PGM sequencer. Data displayed as in c . (e) Scaled chromosomal view of the 5mC MeDIP-Seq enrichment of methylation over the MX1 gene in WT and DKO cells sequenced on the 2 different Ion Torrent sequencers as indicated. MeDIP-Seq data are displayed as RPM (Reads Per Million mapped reads) below the RefSeq annotation of the gene (blue lines). Methylation enrichment is displayed in gray, with red vertical lines corresponding to areas containing CpGs. Chromosome position and gene orientation are displayed.
    Figure Legend Snippet: Scalability of Ion Torrent-compatible MeDIP-Seq protocol. (a) Global 5-methylcytosine (5mC) content measured as a percentage of the genome in DNA from WT and DKO cells determined by an ELISA assay. (b) Number of peaks identified by MACS v2.1.0 software 64 from MeDIP-Seq data in WT and DKO cells. (c) Sequence coverage of genome-wide CpGs for WT and DKO cells on the Ion Torrent Proton sequencer calculated using MEDIPS v1.14.0 software. 39 Pie charts illustrate the fraction of CpGs covered by the indicated reads according to their fold-coverage in relation to the total genomic CpG content from MeDIP-Seq libraries of the indicated sample sequenced on the Proton with the total number of non-redundant mapped reads in parentheses. (d) Sequence coverage of genome-wide CpGs for WT and DKO cells in MeDIP-Seq on the Ion Torrent PGM sequencer. Data displayed as in c . (e) Scaled chromosomal view of the 5mC MeDIP-Seq enrichment of methylation over the MX1 gene in WT and DKO cells sequenced on the 2 different Ion Torrent sequencers as indicated. MeDIP-Seq data are displayed as RPM (Reads Per Million mapped reads) below the RefSeq annotation of the gene (blue lines). Methylation enrichment is displayed in gray, with red vertical lines corresponding to areas containing CpGs. Chromosome position and gene orientation are displayed.

    Techniques Used: Methylated DNA Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Magnetic Cell Separation, Software, Sequencing, Genome Wide, Methylation

    Ion Torrent-compatible MeDIP-Seq accurately distinguishes between variant DNA modifications. (a) Global 5mC (blue bar) content in bulk DNA from a human brain tissue is significantly higher than that of 5hmC (red bar). Percent methylation of the indicated variant modification was measured using an ELISA assay. Mean values of triplicate experiments are shown, with the P -value calculated by 2-sided Student's t -test. (b) Number of peaks identified by MACS v2.1.0 software 64 for 5mC and 5hmC variants of DNA methylation in brain. (c) Venn diagram illustrating the indicated total number of CpGs contained in the peaks identified for 5mC and 5hmC, showing the degree of overlap of CpGs (as a percentage of the total in 5mC and 5hmC) between the 2 libraries. Distribution of 5mC and 5hmC methylation in brain over indicated bp window of all RefSeq annotated TSS (d) , gene bodies (e) , CpG islands (f) , and exons (g) . For d-g , all mean values from brain 5mC (blue line) and 5hmC (red line) are shown as RPM with s.e.m. displayed as a semi-transparent shade around the mean curve. Yellow shaded areas highlight regions of significant difference between 5mC and 5hmC calculated using a one-sided KS test ( P -values shown).
    Figure Legend Snippet: Ion Torrent-compatible MeDIP-Seq accurately distinguishes between variant DNA modifications. (a) Global 5mC (blue bar) content in bulk DNA from a human brain tissue is significantly higher than that of 5hmC (red bar). Percent methylation of the indicated variant modification was measured using an ELISA assay. Mean values of triplicate experiments are shown, with the P -value calculated by 2-sided Student's t -test. (b) Number of peaks identified by MACS v2.1.0 software 64 for 5mC and 5hmC variants of DNA methylation in brain. (c) Venn diagram illustrating the indicated total number of CpGs contained in the peaks identified for 5mC and 5hmC, showing the degree of overlap of CpGs (as a percentage of the total in 5mC and 5hmC) between the 2 libraries. Distribution of 5mC and 5hmC methylation in brain over indicated bp window of all RefSeq annotated TSS (d) , gene bodies (e) , CpG islands (f) , and exons (g) . For d-g , all mean values from brain 5mC (blue line) and 5hmC (red line) are shown as RPM with s.e.m. displayed as a semi-transparent shade around the mean curve. Yellow shaded areas highlight regions of significant difference between 5mC and 5hmC calculated using a one-sided KS test ( P -values shown).

    Techniques Used: Methylated DNA Immunoprecipitation, Variant Assay, Methylation, Modification, Enzyme-linked Immunosorbent Assay, Magnetic Cell Separation, Software, DNA Methylation Assay

    Workflow of Ion Torrent-compatible MeDIP-Seq protocol. Schematic representation of the MeDIP-Seq protocol developed for the Ion Torrent platform to profile DNA methylation genome wide. (a) Genomic DNA (1 μg) is isolated from a sample of interest and sonicated for 300 s on a Covaris Focused-ultrasonicator (Peak intensity power: 50, Duty: 20%, Cycles: 200, Temp: 20°C) to a mean fragment size of 300 bp. (b) Fragmented DNA is nick repaired and ligated with Ion Torrent sequencing adapters. (c) Ligated DNA is enriched for either 5mC (data shown) or 5hmC variants of methylation. Efficiency of MeDIP reaction is determined by using spiked in methylated and unmethylated DNA and compared to a 10% input DNA control. (d) Fragments of immunoprecipitated DNA are separated on a 2% agarose gel and size selected from 200–400 bp. (e) Size-selected fragmented DNA library is amplified for 18 cycles. (f) Amplified MeDIP library is cleaned up and size selected again on a 2% E-gel. (g) MeDIP library (1 μl) is used on a bioanalyzer instrument to assess quality and determine concentration. Figure displays bioanalyzer traces for WT (top) and DKO (bottom) 5mC MeDIP libraries. (h) High quality MeDIP library is templated on ISP beads appropriate to the capacity of sequencing (Life Technologies). (i) Templated MeDIP library is loaded onto an Ion Torrent semi-conductor sequencing chip and sequenced for 500 flows (Life Technologies). An example of the Ion Torrent 318 chip is shown in the figure using scanning electron microscopy to visualize the wells of a semi-conductor sequencing chip. (j) Sequencing data is processed on the Ion Torrent software (Life Technologies). An example of a loaded P1 chip after an Ion Proton sequencing run is shown. WT, HCT116-WT; DKO, HCT116-DKO.
    Figure Legend Snippet: Workflow of Ion Torrent-compatible MeDIP-Seq protocol. Schematic representation of the MeDIP-Seq protocol developed for the Ion Torrent platform to profile DNA methylation genome wide. (a) Genomic DNA (1 μg) is isolated from a sample of interest and sonicated for 300 s on a Covaris Focused-ultrasonicator (Peak intensity power: 50, Duty: 20%, Cycles: 200, Temp: 20°C) to a mean fragment size of 300 bp. (b) Fragmented DNA is nick repaired and ligated with Ion Torrent sequencing adapters. (c) Ligated DNA is enriched for either 5mC (data shown) or 5hmC variants of methylation. Efficiency of MeDIP reaction is determined by using spiked in methylated and unmethylated DNA and compared to a 10% input DNA control. (d) Fragments of immunoprecipitated DNA are separated on a 2% agarose gel and size selected from 200–400 bp. (e) Size-selected fragmented DNA library is amplified for 18 cycles. (f) Amplified MeDIP library is cleaned up and size selected again on a 2% E-gel. (g) MeDIP library (1 μl) is used on a bioanalyzer instrument to assess quality and determine concentration. Figure displays bioanalyzer traces for WT (top) and DKO (bottom) 5mC MeDIP libraries. (h) High quality MeDIP library is templated on ISP beads appropriate to the capacity of sequencing (Life Technologies). (i) Templated MeDIP library is loaded onto an Ion Torrent semi-conductor sequencing chip and sequenced for 500 flows (Life Technologies). An example of the Ion Torrent 318 chip is shown in the figure using scanning electron microscopy to visualize the wells of a semi-conductor sequencing chip. (j) Sequencing data is processed on the Ion Torrent software (Life Technologies). An example of a loaded P1 chip after an Ion Proton sequencing run is shown. WT, HCT116-WT; DKO, HCT116-DKO.

    Techniques Used: Methylated DNA Immunoprecipitation, DNA Methylation Assay, Genome Wide, Isolation, Sonication, Sequencing, Methylation, Immunoprecipitation, Agarose Gel Electrophoresis, Amplification, Concentration Assay, Chromatin Immunoprecipitation, Electron Microscopy, Software

    Ion Torrent-compatible MeDIP-Seq detects expected differences in DNA methylation between DNMT -proficient and - deficient cells. Distribution of 5mC methylation over the indicated bp window of all RefSeq annotated TSS ( a ), gene bodies ( b ), CpG islands ( c ), and exons ( d ). Methylation of WT (green line) and DKO (orange line) HCT116 cells are displayed as mean RPM values with s.e.m. indicated as a semi-transparent shade around the mean curve. Yellow shaded areas highlight regions of significant difference between WT and DKO methylation calculated using a one-sided KS test ( P -values shown). In c and d , a KS test was performed using data over the non-shaded areas as well ( P -values shown).
    Figure Legend Snippet: Ion Torrent-compatible MeDIP-Seq detects expected differences in DNA methylation between DNMT -proficient and - deficient cells. Distribution of 5mC methylation over the indicated bp window of all RefSeq annotated TSS ( a ), gene bodies ( b ), CpG islands ( c ), and exons ( d ). Methylation of WT (green line) and DKO (orange line) HCT116 cells are displayed as mean RPM values with s.e.m. indicated as a semi-transparent shade around the mean curve. Yellow shaded areas highlight regions of significant difference between WT and DKO methylation calculated using a one-sided KS test ( P -values shown). In c and d , a KS test was performed using data over the non-shaded areas as well ( P -values shown).

    Techniques Used: Methylated DNA Immunoprecipitation, DNA Methylation Assay, Methylation

    Ion Torrent-compatible MeDIP-Seq confirms a role for DNA methylation in alternative splicing. (a) Distribution of 5mC methylation over alternatively spliced exons (ASE) flanked by constitutively spliced exons (5’ or 3’ exons) in WT and DKO cells. Mean RPM values are displayed over ± 500 bp windows relative to the splice acceptor and donor sites at each of the exons as indicated, with s.e.m. depicted by shaded areas for WT (green) and DKO (orange) profiles. A schematic representation of cassette exons is shown, with the number of exons aberrantly included (blue) and excluded (gray) in WT vs. DKO cells indicated. P- values shown were calculated using one-sided KS test. (b) The ASE of the FAM204A gene (exon 2) is aberrantly excluded in DKO compared to WT cells. Schematic shows the 2 known isoforms for the FAM204A gene (blue bars depict the first 3 exons of the gene in the orientation indicated), under which a Sashimi plot generated by MISO analysis of RNA-Seq data measured as RPKM in WT and DKO cells illustrates the number of exon-exon junction reads as indicated to infer isoform expression. The left graphs show the MISO calculated distribution of a percent exon inclusion score (Psi-value; 95% confidence intervals in brackets) for the FAM204A ASE from WT (top) and DKO (bottom) RNA-Seq data. (c) Demethylation of the FAM204A ASE correlates with its aberrant exclusion. Scaled chromosomal view at the FAM204A gene region (ASE highlighted in yellow) of the indicated WT and DKO distribution of RNA-Seq (top pair) and DNA methylation (bottom pair) data displayed as RPKM. The first 3 exons of the gene are represented (blue bars) with a CpG island in the promoter region indicated (green bar). In the MeDIP-Seq data, red vertical lines correspond to areas containing CpGs.
    Figure Legend Snippet: Ion Torrent-compatible MeDIP-Seq confirms a role for DNA methylation in alternative splicing. (a) Distribution of 5mC methylation over alternatively spliced exons (ASE) flanked by constitutively spliced exons (5’ or 3’ exons) in WT and DKO cells. Mean RPM values are displayed over ± 500 bp windows relative to the splice acceptor and donor sites at each of the exons as indicated, with s.e.m. depicted by shaded areas for WT (green) and DKO (orange) profiles. A schematic representation of cassette exons is shown, with the number of exons aberrantly included (blue) and excluded (gray) in WT vs. DKO cells indicated. P- values shown were calculated using one-sided KS test. (b) The ASE of the FAM204A gene (exon 2) is aberrantly excluded in DKO compared to WT cells. Schematic shows the 2 known isoforms for the FAM204A gene (blue bars depict the first 3 exons of the gene in the orientation indicated), under which a Sashimi plot generated by MISO analysis of RNA-Seq data measured as RPKM in WT and DKO cells illustrates the number of exon-exon junction reads as indicated to infer isoform expression. The left graphs show the MISO calculated distribution of a percent exon inclusion score (Psi-value; 95% confidence intervals in brackets) for the FAM204A ASE from WT (top) and DKO (bottom) RNA-Seq data. (c) Demethylation of the FAM204A ASE correlates with its aberrant exclusion. Scaled chromosomal view at the FAM204A gene region (ASE highlighted in yellow) of the indicated WT and DKO distribution of RNA-Seq (top pair) and DNA methylation (bottom pair) data displayed as RPKM. The first 3 exons of the gene are represented (blue bars) with a CpG island in the promoter region indicated (green bar). In the MeDIP-Seq data, red vertical lines correspond to areas containing CpGs.

    Techniques Used: Methylated DNA Immunoprecipitation, DNA Methylation Assay, Methylation, Generated, RNA Sequencing Assay, Expressing

    3) Product Images from "Ligation Bias in Illumina Next-Generation DNA Libraries: Implications for Sequencing Ancient Genomes"

    Article Title: Ligation Bias in Illumina Next-Generation DNA Libraries: Implications for Sequencing Ancient Genomes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0078575

    A low adapter concentration magnifies the base composition bias for AT libraries. Aliquots of E. coli DNA extracts were sheared using the Covaris E210 sonicator, size selected, and built into AT libraries using a low (“low”, 0.012 µM) or a standard (“std”, 0.6 µM) adapter concentration. We report the base composition observed at the start (position +1) and the end (position N) of sequences for reads mapping with high quality a unique position of the E. coli NC_010473 genome. The base compositions of the first nucleotide located upstream (position –1) and downstream (position N+1) read alignments are also provided. The average base composition is reported with dashed lines and is calculated using base counts within the reference genome.
    Figure Legend Snippet: A low adapter concentration magnifies the base composition bias for AT libraries. Aliquots of E. coli DNA extracts were sheared using the Covaris E210 sonicator, size selected, and built into AT libraries using a low (“low”, 0.012 µM) or a standard (“std”, 0.6 µM) adapter concentration. We report the base composition observed at the start (position +1) and the end (position N) of sequences for reads mapping with high quality a unique position of the E. coli NC_010473 genome. The base compositions of the first nucleotide located upstream (position –1) and downstream (position N+1) read alignments are also provided. The average base composition is reported with dashed lines and is calculated using base counts within the reference genome.

    Techniques Used: Concentration Assay

    Base composition bias: AT versus BE libraries. Fresh aliquots of E. coli DNA extracts were sheared using the Covaris E210 sonicator, size selected, and built into AT and BE libraries (adapter concentration = 0.6 µM). We report the base composition observed at the first 10 (positions 1 to 10) and last 10 (positions N-9 to N) nucleotide positions within sequence reads mapping with high quality a unique position of the E. coli NC_010473 genome. The genomic composition of the 10 nucleotides located upstream (positions –10 to –1) and downstream (positions N+1 to N+10) DNA inserts are also provided.
    Figure Legend Snippet: Base composition bias: AT versus BE libraries. Fresh aliquots of E. coli DNA extracts were sheared using the Covaris E210 sonicator, size selected, and built into AT and BE libraries (adapter concentration = 0.6 µM). We report the base composition observed at the first 10 (positions 1 to 10) and last 10 (positions N-9 to N) nucleotide positions within sequence reads mapping with high quality a unique position of the E. coli NC_010473 genome. The genomic composition of the 10 nucleotides located upstream (positions –10 to –1) and downstream (positions N+1 to N+10) DNA inserts are also provided.

    Techniques Used: Concentration Assay, Sequencing

    4) 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

    5) Product Images from "Semiconductor-based sequencing of genome-wide DNA methylation states"

    Article Title: Semiconductor-based sequencing of genome-wide DNA methylation states

    Journal: Epigenetics

    doi: 10.1080/15592294.2014.1003747

    Ion Torrent-compatible MeDIP-Seq accurately distinguishes between variant DNA modifications. (a) Global 5mC (blue bar) content in bulk DNA from a human brain tissue is significantly higher than that of 5hmC (red bar). Percent methylation of the indicated variant modification was measured using an ELISA assay. Mean values of triplicate experiments are shown, with the P -value calculated by 2-sided Student's t -test. (b) Number of peaks identified by MACS v2.1.0 software 64 for 5mC and 5hmC variants of DNA methylation in brain. (c) Venn diagram illustrating the indicated total number of CpGs contained in the peaks identified for 5mC and 5hmC, showing the degree of overlap of CpGs (as a percentage of the total in 5mC and 5hmC) between the 2 libraries. Distribution of 5mC and 5hmC methylation in brain over indicated bp window of all RefSeq annotated TSS (d) , gene bodies (e) , CpG islands (f) , and exons (g) . For d-g , all mean values from brain 5mC (blue line) and 5hmC (red line) are shown as RPM with s.e.m. displayed as a semi-transparent shade around the mean curve. Yellow shaded areas highlight regions of significant difference between 5mC and 5hmC calculated using a one-sided KS test ( P -values shown).
    Figure Legend Snippet: Ion Torrent-compatible MeDIP-Seq accurately distinguishes between variant DNA modifications. (a) Global 5mC (blue bar) content in bulk DNA from a human brain tissue is significantly higher than that of 5hmC (red bar). Percent methylation of the indicated variant modification was measured using an ELISA assay. Mean values of triplicate experiments are shown, with the P -value calculated by 2-sided Student's t -test. (b) Number of peaks identified by MACS v2.1.0 software 64 for 5mC and 5hmC variants of DNA methylation in brain. (c) Venn diagram illustrating the indicated total number of CpGs contained in the peaks identified for 5mC and 5hmC, showing the degree of overlap of CpGs (as a percentage of the total in 5mC and 5hmC) between the 2 libraries. Distribution of 5mC and 5hmC methylation in brain over indicated bp window of all RefSeq annotated TSS (d) , gene bodies (e) , CpG islands (f) , and exons (g) . For d-g , all mean values from brain 5mC (blue line) and 5hmC (red line) are shown as RPM with s.e.m. displayed as a semi-transparent shade around the mean curve. Yellow shaded areas highlight regions of significant difference between 5mC and 5hmC calculated using a one-sided KS test ( P -values shown).

    Techniques Used: Methylated DNA Immunoprecipitation, Variant Assay, Methylation, Modification, Enzyme-linked Immunosorbent Assay, Magnetic Cell Separation, Software, DNA Methylation Assay

    Workflow of Ion Torrent-compatible MeDIP-Seq protocol. Schematic representation of the MeDIP-Seq protocol developed for the Ion Torrent platform to profile DNA methylation genome wide. (a) Genomic DNA (1 μg) is isolated from a sample of interest and sonicated for 300 s on a Covaris Focused-ultrasonicator (Peak intensity power: 50, Duty: 20%, Cycles: 200, Temp: 20°C) to a mean fragment size of 300 bp. (b) Fragmented DNA is nick repaired and ligated with Ion Torrent sequencing adapters. (c) Ligated DNA is enriched for either 5mC (data shown) or 5hmC variants of methylation. Efficiency of MeDIP reaction is determined by using spiked in methylated and unmethylated DNA and compared to a 10% input DNA control. (d) Fragments of immunoprecipitated DNA are separated on a 2% agarose gel and size selected from 200–400 bp. (e) Size-selected fragmented DNA library is amplified for 18 cycles. (f) Amplified MeDIP library is cleaned up and size selected again on a 2% E-gel. (g) MeDIP library (1 μl) is used on a bioanalyzer instrument to assess quality and determine concentration. Figure displays bioanalyzer traces for WT (top) and DKO (bottom) 5mC MeDIP libraries. (h) High quality MeDIP library is templated on ISP beads appropriate to the capacity of sequencing (Life Technologies). (i) Templated MeDIP library is loaded onto an Ion Torrent semi-conductor sequencing chip and sequenced for 500 flows (Life Technologies). An example of the Ion Torrent 318 chip is shown in the figure using scanning electron microscopy to visualize the wells of a semi-conductor sequencing chip. (j) Sequencing data is processed on the Ion Torrent software (Life Technologies). An example of a loaded P1 chip after an Ion Proton sequencing run is shown. WT, HCT116-WT; DKO, HCT116-DKO.
    Figure Legend Snippet: Workflow of Ion Torrent-compatible MeDIP-Seq protocol. Schematic representation of the MeDIP-Seq protocol developed for the Ion Torrent platform to profile DNA methylation genome wide. (a) Genomic DNA (1 μg) is isolated from a sample of interest and sonicated for 300 s on a Covaris Focused-ultrasonicator (Peak intensity power: 50, Duty: 20%, Cycles: 200, Temp: 20°C) to a mean fragment size of 300 bp. (b) Fragmented DNA is nick repaired and ligated with Ion Torrent sequencing adapters. (c) Ligated DNA is enriched for either 5mC (data shown) or 5hmC variants of methylation. Efficiency of MeDIP reaction is determined by using spiked in methylated and unmethylated DNA and compared to a 10% input DNA control. (d) Fragments of immunoprecipitated DNA are separated on a 2% agarose gel and size selected from 200–400 bp. (e) Size-selected fragmented DNA library is amplified for 18 cycles. (f) Amplified MeDIP library is cleaned up and size selected again on a 2% E-gel. (g) MeDIP library (1 μl) is used on a bioanalyzer instrument to assess quality and determine concentration. Figure displays bioanalyzer traces for WT (top) and DKO (bottom) 5mC MeDIP libraries. (h) High quality MeDIP library is templated on ISP beads appropriate to the capacity of sequencing (Life Technologies). (i) Templated MeDIP library is loaded onto an Ion Torrent semi-conductor sequencing chip and sequenced for 500 flows (Life Technologies). An example of the Ion Torrent 318 chip is shown in the figure using scanning electron microscopy to visualize the wells of a semi-conductor sequencing chip. (j) Sequencing data is processed on the Ion Torrent software (Life Technologies). An example of a loaded P1 chip after an Ion Proton sequencing run is shown. WT, HCT116-WT; DKO, HCT116-DKO.

    Techniques Used: Methylated DNA Immunoprecipitation, DNA Methylation Assay, Genome Wide, Isolation, Sonication, Sequencing, Methylation, Immunoprecipitation, Agarose Gel Electrophoresis, Amplification, Concentration Assay, Chromatin Immunoprecipitation, Electron Microscopy, Software

    6) 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

    Related Articles

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    Article Title: Integrated genomic characterization of oesophageal carcinoma
    Article Snippet: .. Low-pass whole-genome sequencing for rearrangement identification Genomic DNA (500–700 ng per sample) was sheared into 250-bp fragments using a Covaris E220 ultrasonicator, then converted to a paired-end Illumina library using KAPA Bio kits with Caliper (PerkinElmer) robotic NGS Suite (Partek Genomics) according to manufacturers’ protocols. ..

    Sonication:

    Article Title: Assessing the Impact of Cyclosporin A on Lentiviral Transduction and Preservation of Human Hematopoietic Stem Cells in Clinically Relevant Ex Vivo Gene Therapy Settings
    Article Snippet: .. Briefly, the genomic DNA from transduced cells was fragmented by adaptive focused acoustic sonication using a Covaris E220 Ultrasonicator (Covaris, Inc., Woburn, MA). .. The fragmented DNA was then subjected to end repair and 3′ adenylation, ligated to the DNA blunted-end linker cassette using the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® following the manufacturer's instructions (New England Biolabs, Ipswich, MA).

    Article Title: An RNA-Centric Dissection of Host Complexes Controlling Flavivirus Infection
    Article Snippet: .. Lysates were sonicated using a focused-ultrasonicator (Covaris, E220) until the average RNA length was 500 nucleotides as determined by agarose gel analysis and stored at −80°C. .. Stored lysates were thawed on ice, and for every 1 mL of sonicated lysate 2 mL of ChIRP hybridization buffer (750 mM NaCl, 1% SDS, 50 mM Tris-HCl pH 7.0, 1 mM EDTA, 15% formamide; made fresh) and precleared by adding 30 μL washed MyOne C1 beads per mL of lysate at 37°C for 30 minutes on rotation.

    Article Title: Pluripotency transcription factors and Tet1/2 maintain Brd4-independent stem cell identity
    Article Snippet: .. For Med1 and Nanog IP, cells were resuspended in 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA, 0.1% SDS and 1% Triton X-100 and sonicated for approximately 8 min using a Covaris E220 ultrasonicator. .. The resulting whole cell extract was centrifuged and the supernatant was incubated for 1 h at 4 °C with 50 μL of protein G magnetic beads that had been pre-incubated with 5 μg of control rabbit IgG.

    Next-Generation Sequencing:

    Article Title: Integrated genomic characterization of oesophageal carcinoma
    Article Snippet: .. Low-pass whole-genome sequencing for rearrangement identification Genomic DNA (500–700 ng per sample) was sheared into 250-bp fragments using a Covaris E220 ultrasonicator, then converted to a paired-end Illumina library using KAPA Bio kits with Caliper (PerkinElmer) robotic NGS Suite (Partek Genomics) according to manufacturers’ protocols. ..

    Agarose Gel Electrophoresis:

    Article Title: An RNA-Centric Dissection of Host Complexes Controlling Flavivirus Infection
    Article Snippet: .. Lysates were sonicated using a focused-ultrasonicator (Covaris, E220) until the average RNA length was 500 nucleotides as determined by agarose gel analysis and stored at −80°C. .. Stored lysates were thawed on ice, and for every 1 mL of sonicated lysate 2 mL of ChIRP hybridization buffer (750 mM NaCl, 1% SDS, 50 mM Tris-HCl pH 7.0, 1 mM EDTA, 15% formamide; made fresh) and precleared by adding 30 μL washed MyOne C1 beads per mL of lysate at 37°C for 30 minutes on rotation.

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  • 90
    Covaris microtube 50
    Microtube 50, supplied by Covaris, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/microtube 50/product/Covaris
    Average 90 stars, based on 3 article reviews
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    microtube 50 - by Bioz Stars, 2020-05
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    92
    Covaris dna samples
    Dna Samples, supplied by Covaris, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Covaris microtube afa fiber screw cap
    Microtube Afa Fiber Screw Cap, supplied by Covaris, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 2 article reviews
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    91
    Covaris microtubes
    Microtubes, supplied by Covaris, used in various techniques. Bioz Stars score: 91/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/microtubes/product/Covaris
    Average 91 stars, based on 22 article reviews
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