cviki 1  (New England Biolabs)


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    Name:
    CviKI 1
    Description:
    CviKI 1 250 units
    Catalog Number:
    r0710l
    Price:
    70
    Size:
    250 units
    Category:
    Restriction Enzymes
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    New England Biolabs cviki 1
    CviKI 1
    CviKI 1 250 units
    https://www.bioz.com/result/cviki 1/product/New England Biolabs
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    cviki 1 - by Bioz Stars, 2020-04
    99/100 stars

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    1) Product Images from "RELACS nuclei barcoding enables high-throughput ChIP-seq"

    Article Title: RELACS nuclei barcoding enables high-throughput ChIP-seq

    Journal: Communications Biology

    doi: 10.1038/s42003-018-0219-z

    RELACS workflow. Overview of the RELACS method. The protocol facilitates barcoding multiple cell populations, which can be pooled and investigated for multiple epitopes within the same run. The method starts by isolating nuclei from a pool of formaldehyde-fixed cells, using sonication to reduce cell type dependency 5 ( a ). The nuclear membrane is permeabilized to allow entrance of enzymes, followed by DNA barcodes. Restriction endonucleases with a high frequency of recognition sites are used to fragment chromatin—in this work CviKI-1 was used ( b ). Nuclei are washed to remove active restriction enzymes ( c ). Hairpin adapters harboring barcodes are ligated to both the ends of the fragmented chromatin inside the nuclei. The barcoding has been tested using 100–500,000 nuclei without the need to change protocol conditions ( d ). Cell populations marked with specific barcodes are pooled ( e ), concentrated and lysed using SDS at low concentration and short sonication to release chromatin into solution ( f ). Chromatin is split and incubated with the antibodies of interest ( g ). After ChIP washes and DNA purification (not illustrated), only DNA that harbors nuclei barcodes at both ends is PCR amplified to complete library construction. PCR amplification appends an Illumina barcode onto mark each fragment ( h ). Sequenced libraries are demultiplexed by Illumina barcode, to retrieve ChIP information, and then nuclear barcode, to identify the initial cell population ( i ). The RELACS protocol is very fast and ChIP-seq libraries can be generated for hundreds of samples within three days
    Figure Legend Snippet: RELACS workflow. Overview of the RELACS method. The protocol facilitates barcoding multiple cell populations, which can be pooled and investigated for multiple epitopes within the same run. The method starts by isolating nuclei from a pool of formaldehyde-fixed cells, using sonication to reduce cell type dependency 5 ( a ). The nuclear membrane is permeabilized to allow entrance of enzymes, followed by DNA barcodes. Restriction endonucleases with a high frequency of recognition sites are used to fragment chromatin—in this work CviKI-1 was used ( b ). Nuclei are washed to remove active restriction enzymes ( c ). Hairpin adapters harboring barcodes are ligated to both the ends of the fragmented chromatin inside the nuclei. The barcoding has been tested using 100–500,000 nuclei without the need to change protocol conditions ( d ). Cell populations marked with specific barcodes are pooled ( e ), concentrated and lysed using SDS at low concentration and short sonication to release chromatin into solution ( f ). Chromatin is split and incubated with the antibodies of interest ( g ). After ChIP washes and DNA purification (not illustrated), only DNA that harbors nuclei barcodes at both ends is PCR amplified to complete library construction. PCR amplification appends an Illumina barcode onto mark each fragment ( h ). Sequenced libraries are demultiplexed by Illumina barcode, to retrieve ChIP information, and then nuclear barcode, to identify the initial cell population ( i ). The RELACS protocol is very fast and ChIP-seq libraries can be generated for hundreds of samples within three days

    Techniques Used: Sonication, Concentration Assay, Incubation, Chromatin Immunoprecipitation, DNA Purification, Polymerase Chain Reaction, Amplification, Generated

    Related Articles

    Clone Assay:

    Article Title: Application of M13 phage display for identifying immunogenic proteins from tick (Ixodes scapularis) saliva
    Article Snippet: .. Salivary gland library construction To generate blunt ended DNA fragments suitable for cloning into pHORF3 [ ] a restriction enzyme digest using the blunt end cutting enzymes AfeI, AluI and CviKI-1 (NEB, Frankfurt, Germany) was performed. .. 30 μl SMART cDNA (~1.7 μg) were mixed with 1.25 U of each of the restriction enzymes, 10 μL NEBuffer 4 (NEB), 10 μL 10xBSA solution (NEB) in a total volume of 100 μl.

    Amplification:

    Article Title: The -258 A/G (SNP rs12885300) polymorphism of the human type-2 deiodinase gene is associated with a shift in the pattern of secretion of thyroid hormones following a TRH-induced acute rise in TSH
    Article Snippet: The −258 A/G DIO2 polymorphism status was characterized using established methods by PCR restriction fragmente amplified with the following primers: forward 5'-AAAGCTGGCGTACTCGTC-3', and reverse 5'-AAAGAGCATAGAGACAATGAAAG-3'. .. After purification, PCR products were digested with CviKI-1 (New England BioLabs, Ipswich, MA), restriction enzyme for 4 hours at 37°C.

    Article Title: Complete chloroplast genome sequence and phylogenetic analysis of wasabi (Eutrema japonicum) and its relatives
    Article Snippet: An additional primer set for the ribulose bisphosphate carboxylase large chain gene (rbcL ) in the cp genome was used as the positive control amplicon, as described . .. The PCR products were digested at two or three sites by the restriction enzyme, CviKI -1 (New England Biolabs, Ipswich, MA).

    Positive Control:

    Article Title: Complete chloroplast genome sequence and phylogenetic analysis of wasabi (Eutrema japonicum) and its relatives
    Article Snippet: An additional primer set for the ribulose bisphosphate carboxylase large chain gene (rbcL ) in the cp genome was used as the positive control amplicon, as described . .. The PCR products were digested at two or three sites by the restriction enzyme, CviKI -1 (New England Biolabs, Ipswich, MA).

    Construct:

    Article Title: SMASH, a fragmentation and sequencing method for genomic copy number analysis
    Article Snippet: We tested three different genomic DNA inputs—200 ng, 500 ng, and 1 µg—and successfully constructed high-quality libraries for all three conditions. .. DNA fragments were further cut by CviKI-1 (NEB) and NlaIII (NEB) in 1× CutSmart buffer in a final volume of 90 µL, which was incubated at 37°C for 1 h. After enzymatic digestion, the solution volume was reduced to ∼30 µL by Savant SpeedVac (Thermo Scientific).

    Article Title: Complete chloroplast genome sequence and phylogenetic analysis of wasabi (Eutrema japonicum) and its relatives
    Article Snippet: The PCR products were digested at two or three sites by the restriction enzyme, CviKI -1 (New England Biolabs, Ipswich, MA). .. Only two polymorphic sites were detected between ‘Shimane No. 3’ and the other accessions, therefore a multiplex PCR marker was constructed within the coding region of the rpoC2 gene, using specific primer pairs.

    Electrophoresis:

    Article Title: The -258 A/G (SNP rs12885300) polymorphism of the human type-2 deiodinase gene is associated with a shift in the pattern of secretion of thyroid hormones following a TRH-induced acute rise in TSH
    Article Snippet: After purification, PCR products were digested with CviKI-1 (New England BioLabs, Ipswich, MA), restriction enzyme for 4 hours at 37°C. .. The digestion products were separated on 4.5% agarose gel by electrophoresis and visualized by ethidium bromide staining.

    Article Title: Population-Based Molecular Epidemiology of Leprosy in Cebu, Philippines
    Article Snippet: Restriction digest assays were developed using SmlI, CviKI-1, and BstUI for SNP loci 1, 2, and 3, respectively (New England Biolabs, MA). .. The SmlI, BstUI, and CviKI-1 reactions were performed at 55°C, 60°C, and 37°C, respectively, for 1 h. The SmlI- and BstUI-uncut and -cut DNAs were subjected to electrophoresis on 3% agarose gels.

    Incubation:

    Article Title: SMASH, a fragmentation and sequencing method for genomic copy number analysis
    Article Snippet: .. DNA fragments were further cut by CviKI-1 (NEB) and NlaIII (NEB) in 1× CutSmart buffer in a final volume of 90 µL, which was incubated at 37°C for 1 h. After enzymatic digestion, the solution volume was reduced to ∼30 µL by Savant SpeedVac (Thermo Scientific). .. DNA fragments > 100 bp were removed as follows: adding 2.5× volume of AMPure XP beads (Beckman Coulter), mixing well, incubating at room temperature (RT) for 5 min, and collecting the supernatant.

    Article Title: Application of M13 phage display for identifying immunogenic proteins from tick (Ixodes scapularis) saliva
    Article Snippet: Salivary gland library construction To generate blunt ended DNA fragments suitable for cloning into pHORF3 [ ] a restriction enzyme digest using the blunt end cutting enzymes AfeI, AluI and CviKI-1 (NEB, Frankfurt, Germany) was performed. .. The digestion was incubated at 37 °C for 10 min followed by a heat inactivation at 65 °C for 20 min.

    Activity Assay:

    Article Title: RELACS nuclei barcoding enables high-throughput ChIP-seq
    Article Snippet: Nuclei digestion Five units of the restriction enzyme CviKI-1 (NEB, R0710S) were added to every 100,000 nuclei. .. Note that the restriction endonucleases (used in RELACS) lack exonuclease activity and are easier to titrate upon variation of the input material.

    Acrylamide Gel Assay:

    Article Title: Population-Based Molecular Epidemiology of Leprosy in Cebu, Philippines
    Article Snippet: Restriction digest assays were developed using SmlI, CviKI-1, and BstUI for SNP loci 1, 2, and 3, respectively (New England Biolabs, MA). .. The uncut and CviKI-1-digested SNP locus 2 products were resolved on a 12% acrylamide gel.

    Transformation Assay:

    Article Title: A genome-wide inducible phenotypic screen identifies antisense RNA constructs silencing Escherichia coli essential genes
    Article Snippet: Genomic DNA was extracted from E. coli MG1655 cells (American Type Culture Collection, Manassas, VA) using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI) followed by partial digest with Sau 3AI or Cvi KI-1 (NEB, Ipswich, MA). .. Ligation mixtures were transformed into E. coli DH5α competent cells (Life Technologies) and plated onto LB agar plates plus 34 μg mL−1 chloramphenicol.

    Ligation:

    Article Title: A genome-wide inducible phenotypic screen identifies antisense RNA constructs silencing Escherichia coli essential genes
    Article Snippet: Genomic DNA was extracted from E. coli MG1655 cells (American Type Culture Collection, Manassas, VA) using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI) followed by partial digest with Sau 3AI or Cvi KI-1 (NEB, Ipswich, MA). .. Ligation mixtures were transformed into E. coli DH5α competent cells (Life Technologies) and plated onto LB agar plates plus 34 μg mL−1 chloramphenicol.

    Generated:

    Article Title: Complete chloroplast genome sequence and phylogenetic analysis of wasabi (Eutrema japonicum) and its relatives
    Article Snippet: The PCR products were digested at two or three sites by the restriction enzyme, CviKI -1 (New England Biolabs, Ipswich, MA). .. As expected from the cp nucleotide sequences, three bands (227, 386, and 407 bp) were generated in ‘Fujidaruma’ and ‘Shimane No. 3’, whereas three bands of 227, 350 and 386 bp in size were detected in the other cultivated accessions (Fig. ).

    Polymerase Chain Reaction:

    Article Title: The -258 A/G (SNP rs12885300) polymorphism of the human type-2 deiodinase gene is associated with a shift in the pattern of secretion of thyroid hormones following a TRH-induced acute rise in TSH
    Article Snippet: .. After purification, PCR products were digested with CviKI-1 (New England BioLabs, Ipswich, MA), restriction enzyme for 4 hours at 37°C. .. The digestion products were separated on 4.5% agarose gel by electrophoresis and visualized by ethidium bromide staining.

    Article Title: Complete chloroplast genome sequence and phylogenetic analysis of wasabi (Eutrema japonicum) and its relatives
    Article Snippet: .. The PCR products were digested at two or three sites by the restriction enzyme, CviKI -1 (New England Biolabs, Ipswich, MA). .. As expected from the cp nucleotide sequences, three bands (227, 386, and 407 bp) were generated in ‘Fujidaruma’ and ‘Shimane No. 3’, whereas three bands of 227, 350 and 386 bp in size were detected in the other cultivated accessions (Fig. ).

    Article Title: Population-Based Molecular Epidemiology of Leprosy in Cebu, Philippines
    Article Snippet: Paragraph title: Development of a PCR-RFLP method for SNP subtyping of M. leprae . ... Restriction digest assays were developed using SmlI, CviKI-1, and BstUI for SNP loci 1, 2, and 3, respectively (New England Biolabs, MA).

    DNA Extraction:

    Article Title: The -258 A/G (SNP rs12885300) polymorphism of the human type-2 deiodinase gene is associated with a shift in the pattern of secretion of thyroid hormones following a TRH-induced acute rise in TSH
    Article Snippet: Paragraph title: DNA isolation and DIO2 restriction fragment polymorphisms analysis ... After purification, PCR products were digested with CviKI-1 (New England BioLabs, Ipswich, MA), restriction enzyme for 4 hours at 37°C.

    Isolation:

    Article Title: The -258 A/G (SNP rs12885300) polymorphism of the human type-2 deiodinase gene is associated with a shift in the pattern of secretion of thyroid hormones following a TRH-induced acute rise in TSH
    Article Snippet: After isolation, DNA concentration was measured using a NanoDrop™ spectrophotometer (Thermo Fisher Scientific, Waltham, MA). .. After purification, PCR products were digested with CviKI-1 (New England BioLabs, Ipswich, MA), restriction enzyme for 4 hours at 37°C.

    Purification:

    Article Title: SMASH, a fragmentation and sequencing method for genomic copy number analysis
    Article Snippet: DNA fragments were further cut by CviKI-1 (NEB) and NlaIII (NEB) in 1× CutSmart buffer in a final volume of 90 µL, which was incubated at 37°C for 1 h. After enzymatic digestion, the solution volume was reduced to ∼30 µL by Savant SpeedVac (Thermo Scientific). .. The supernatant was then purified by QIAquick nucleotide removal kit (Qiagen), following the manufacturer's instructions.

    Article Title: A genome-wide inducible phenotypic screen identifies antisense RNA constructs silencing Escherichia coli essential genes
    Article Snippet: Genomic DNA was extracted from E. coli MG1655 cells (American Type Culture Collection, Manassas, VA) using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI) followed by partial digest with Sau 3AI or Cvi KI-1 (NEB, Ipswich, MA). .. The resulting DNA fragments (200–800 bp) were purified from agarose gels using the Zymoclean Gel DNA Recovery Kit (Zymo Research, Orange, CA).

    Article Title: The -258 A/G (SNP rs12885300) polymorphism of the human type-2 deiodinase gene is associated with a shift in the pattern of secretion of thyroid hormones following a TRH-induced acute rise in TSH
    Article Snippet: .. After purification, PCR products were digested with CviKI-1 (New England BioLabs, Ipswich, MA), restriction enzyme for 4 hours at 37°C. .. The digestion products were separated on 4.5% agarose gel by electrophoresis and visualized by ethidium bromide staining.

    Plasmid Preparation:

    Article Title: Application of M13 phage display for identifying immunogenic proteins from tick (Ixodes scapularis) saliva
    Article Snippet: Salivary gland library construction To generate blunt ended DNA fragments suitable for cloning into pHORF3 [ ] a restriction enzyme digest using the blunt end cutting enzymes AfeI, AluI and CviKI-1 (NEB, Frankfurt, Germany) was performed. .. The phagemid vector pHORF3 was linearized by digestion with PmeI (NEB).

    Article Title: A genome-wide inducible phenotypic screen identifies antisense RNA constructs silencing Escherichia coli essential genes
    Article Snippet: Genomic DNA was extracted from E. coli MG1655 cells (American Type Culture Collection, Manassas, VA) using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI) followed by partial digest with Sau 3AI or Cvi KI-1 (NEB, Ipswich, MA). .. Plasmid vector was digested with Bam HI (if Sau 3AI was used to digest the genomic DNA) or Sna BI (if Cvi KI-1 was used), dephosphorylated using Antarctic Phosphatase (NEB) and then ligated with the inserts using the T4 DNA ligase (Life Technologies, Carlsbad, CA).

    Multiplex Assay:

    Article Title: Complete chloroplast genome sequence and phylogenetic analysis of wasabi (Eutrema japonicum) and its relatives
    Article Snippet: The PCR products were digested at two or three sites by the restriction enzyme, CviKI -1 (New England Biolabs, Ipswich, MA). .. Only two polymorphic sites were detected between ‘Shimane No. 3’ and the other accessions, therefore a multiplex PCR marker was constructed within the coding region of the rpoC2 gene, using specific primer pairs.

    Article Title: Population-Based Molecular Epidemiology of Leprosy in Cebu, Philippines
    Article Snippet: Single or multiplex PCRs were set up using 1 to 2 μl of the DNA extract. .. Restriction digest assays were developed using SmlI, CviKI-1, and BstUI for SNP loci 1, 2, and 3, respectively (New England Biolabs, MA).

    Agarose Gel Electrophoresis:

    Article Title: The -258 A/G (SNP rs12885300) polymorphism of the human type-2 deiodinase gene is associated with a shift in the pattern of secretion of thyroid hormones following a TRH-induced acute rise in TSH
    Article Snippet: After purification, PCR products were digested with CviKI-1 (New England BioLabs, Ipswich, MA), restriction enzyme for 4 hours at 37°C. .. The digestion products were separated on 4.5% agarose gel by electrophoresis and visualized by ethidium bromide staining.

    Article Title: Population-Based Molecular Epidemiology of Leprosy in Cebu, Philippines
    Article Snippet: The DNA products were resolved by agarose gel electrophoresis and detected by ethidium bromide staining. .. Restriction digest assays were developed using SmlI, CviKI-1, and BstUI for SNP loci 1, 2, and 3, respectively (New England Biolabs, MA).

    Spectrophotometry:

    Article Title: The -258 A/G (SNP rs12885300) polymorphism of the human type-2 deiodinase gene is associated with a shift in the pattern of secretion of thyroid hormones following a TRH-induced acute rise in TSH
    Article Snippet: After isolation, DNA concentration was measured using a NanoDrop™ spectrophotometer (Thermo Fisher Scientific, Waltham, MA). .. After purification, PCR products were digested with CviKI-1 (New England BioLabs, Ipswich, MA), restriction enzyme for 4 hours at 37°C.

    Concentration Assay:

    Article Title: The -258 A/G (SNP rs12885300) polymorphism of the human type-2 deiodinase gene is associated with a shift in the pattern of secretion of thyroid hormones following a TRH-induced acute rise in TSH
    Article Snippet: After isolation, DNA concentration was measured using a NanoDrop™ spectrophotometer (Thermo Fisher Scientific, Waltham, MA). .. After purification, PCR products were digested with CviKI-1 (New England BioLabs, Ipswich, MA), restriction enzyme for 4 hours at 37°C.

    DNA Purification:

    Article Title: A genome-wide inducible phenotypic screen identifies antisense RNA constructs silencing Escherichia coli essential genes
    Article Snippet: .. Genomic DNA was extracted from E. coli MG1655 cells (American Type Culture Collection, Manassas, VA) using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI) followed by partial digest with Sau 3AI or Cvi KI-1 (NEB, Ipswich, MA). .. The resulting DNA fragments (200–800 bp) were purified from agarose gels using the Zymoclean Gel DNA Recovery Kit (Zymo Research, Orange, CA).

    Marker:

    Article Title: Complete chloroplast genome sequence and phylogenetic analysis of wasabi (Eutrema japonicum) and its relatives
    Article Snippet: The PCR products were digested at two or three sites by the restriction enzyme, CviKI -1 (New England Biolabs, Ipswich, MA). .. Only two polymorphic sites were detected between ‘Shimane No. 3’ and the other accessions, therefore a multiplex PCR marker was constructed within the coding region of the rpoC2 gene, using specific primer pairs.

    Staining:

    Article Title: The -258 A/G (SNP rs12885300) polymorphism of the human type-2 deiodinase gene is associated with a shift in the pattern of secretion of thyroid hormones following a TRH-induced acute rise in TSH
    Article Snippet: After purification, PCR products were digested with CviKI-1 (New England BioLabs, Ipswich, MA), restriction enzyme for 4 hours at 37°C. .. The digestion products were separated on 4.5% agarose gel by electrophoresis and visualized by ethidium bromide staining.

    Article Title: Population-Based Molecular Epidemiology of Leprosy in Cebu, Philippines
    Article Snippet: The DNA products were resolved by agarose gel electrophoresis and detected by ethidium bromide staining. .. Restriction digest assays were developed using SmlI, CviKI-1, and BstUI for SNP loci 1, 2, and 3, respectively (New England Biolabs, MA).

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    New England Biolabs cviki 1
    RELACS workflow. Overview of the RELACS method. The protocol facilitates barcoding multiple cell populations, which can be pooled and investigated for multiple epitopes within the same run. The method starts by isolating nuclei from a pool of formaldehyde-fixed cells, using sonication to reduce cell type dependency 5 ( a ). The nuclear membrane is permeabilized to allow entrance of enzymes, followed by DNA barcodes. Restriction endonucleases with a high frequency of recognition sites are used to fragment chromatin—in this work <t>CviKI-1</t> was used ( b ). Nuclei are washed to remove active restriction enzymes ( c ). Hairpin adapters harboring barcodes are ligated to both the ends of the fragmented chromatin inside the nuclei. The barcoding has been tested using 100–500,000 nuclei without the need to change protocol conditions ( d ). Cell populations marked with specific barcodes are pooled ( e ), concentrated and lysed using SDS at low concentration and short sonication to release chromatin into solution ( f ). Chromatin is split and incubated with the antibodies of interest ( g ). After ChIP washes and DNA purification (not illustrated), only DNA that harbors nuclei barcodes at both ends is PCR amplified to complete library construction. PCR amplification appends an Illumina barcode onto mark each fragment ( h ). Sequenced libraries are demultiplexed by Illumina barcode, to retrieve ChIP information, and then nuclear barcode, to identify the initial cell population ( i ). The RELACS protocol is very fast and ChIP-seq libraries can be generated for hundreds of samples within three days
    Cviki 1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cviki 1/product/New England Biolabs
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    cviki 1 - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

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    RELACS workflow. Overview of the RELACS method. The protocol facilitates barcoding multiple cell populations, which can be pooled and investigated for multiple epitopes within the same run. The method starts by isolating nuclei from a pool of formaldehyde-fixed cells, using sonication to reduce cell type dependency 5 ( a ). The nuclear membrane is permeabilized to allow entrance of enzymes, followed by DNA barcodes. Restriction endonucleases with a high frequency of recognition sites are used to fragment chromatin—in this work CviKI-1 was used ( b ). Nuclei are washed to remove active restriction enzymes ( c ). Hairpin adapters harboring barcodes are ligated to both the ends of the fragmented chromatin inside the nuclei. The barcoding has been tested using 100–500,000 nuclei without the need to change protocol conditions ( d ). Cell populations marked with specific barcodes are pooled ( e ), concentrated and lysed using SDS at low concentration and short sonication to release chromatin into solution ( f ). Chromatin is split and incubated with the antibodies of interest ( g ). After ChIP washes and DNA purification (not illustrated), only DNA that harbors nuclei barcodes at both ends is PCR amplified to complete library construction. PCR amplification appends an Illumina barcode onto mark each fragment ( h ). Sequenced libraries are demultiplexed by Illumina barcode, to retrieve ChIP information, and then nuclear barcode, to identify the initial cell population ( i ). The RELACS protocol is very fast and ChIP-seq libraries can be generated for hundreds of samples within three days

    Journal: Communications Biology

    Article Title: RELACS nuclei barcoding enables high-throughput ChIP-seq

    doi: 10.1038/s42003-018-0219-z

    Figure Lengend Snippet: RELACS workflow. Overview of the RELACS method. The protocol facilitates barcoding multiple cell populations, which can be pooled and investigated for multiple epitopes within the same run. The method starts by isolating nuclei from a pool of formaldehyde-fixed cells, using sonication to reduce cell type dependency 5 ( a ). The nuclear membrane is permeabilized to allow entrance of enzymes, followed by DNA barcodes. Restriction endonucleases with a high frequency of recognition sites are used to fragment chromatin—in this work CviKI-1 was used ( b ). Nuclei are washed to remove active restriction enzymes ( c ). Hairpin adapters harboring barcodes are ligated to both the ends of the fragmented chromatin inside the nuclei. The barcoding has been tested using 100–500,000 nuclei without the need to change protocol conditions ( d ). Cell populations marked with specific barcodes are pooled ( e ), concentrated and lysed using SDS at low concentration and short sonication to release chromatin into solution ( f ). Chromatin is split and incubated with the antibodies of interest ( g ). After ChIP washes and DNA purification (not illustrated), only DNA that harbors nuclei barcodes at both ends is PCR amplified to complete library construction. PCR amplification appends an Illumina barcode onto mark each fragment ( h ). Sequenced libraries are demultiplexed by Illumina barcode, to retrieve ChIP information, and then nuclear barcode, to identify the initial cell population ( i ). The RELACS protocol is very fast and ChIP-seq libraries can be generated for hundreds of samples within three days

    Article Snippet: Nuclei digestion Five units of the restriction enzyme CviKI-1 (NEB, R0710S) were added to every 100,000 nuclei.

    Techniques: Sonication, Concentration Assay, Incubation, Chromatin Immunoprecipitation, DNA Purification, Polymerase Chain Reaction, Amplification, Generated