hifi dna assembly  (New England Biolabs)


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    Name:
    NEBuilder HiFi DNA Assembly Master Mix
    Description:
    NEBuilder HiFi DNA Assembly Master Mix 50 rxns
    Catalog Number:
    e2621l
    Price:
    630
    Size:
    50 rxns
    Category:
    Cloning and Expression Systems
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    Structured Review

    New England Biolabs hifi dna assembly
    NEBuilder HiFi DNA Assembly Master Mix
    NEBuilder HiFi DNA Assembly Master Mix 50 rxns
    https://www.bioz.com/result/hifi dna assembly/product/New England Biolabs
    Average 99 stars, based on 57 article reviews
    Price from $9.99 to $1999.99
    hifi dna assembly - by Bioz Stars, 2021-02
    99/100 stars

    Images

    1) Product Images from "Rational Design of High-Number dsDNA Fragments Based on Thermodynamics for the Construction of Full-Length Genes in a Single Reaction"

    Article Title: Rational Design of High-Number dsDNA Fragments Based on Thermodynamics for the Construction of Full-Length Genes in a Single Reaction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0145682

    Agarose gel electrophoresis of the three assembled genes (a) The first lane contains the GFP gene assembled from 27 dsDNA fragments showing up at the expected 757 bp length. The second lane contains the kanamycin resistance gene assembled from 28 dsDNA fragments showing up at the expected 953 bp length. The third lane contains the tetracycline resistance gene assembled from 45 dsDNA fragments at the expected 1254 bp length. All assemblies were performed using Gibson Assembly master mix. (b) The same assemblies performed using the NEBuilder HiFi DNA Assembly master mix. The agarose gel is stained with ethidium bromide.
    Figure Legend Snippet: Agarose gel electrophoresis of the three assembled genes (a) The first lane contains the GFP gene assembled from 27 dsDNA fragments showing up at the expected 757 bp length. The second lane contains the kanamycin resistance gene assembled from 28 dsDNA fragments showing up at the expected 953 bp length. The third lane contains the tetracycline resistance gene assembled from 45 dsDNA fragments at the expected 1254 bp length. All assemblies were performed using Gibson Assembly master mix. (b) The same assemblies performed using the NEBuilder HiFi DNA Assembly master mix. The agarose gel is stained with ethidium bromide.

    Techniques Used: Agarose Gel Electrophoresis, Staining

    Related Articles

    Construct:

    Article Title: Mosaic Ends Tagmentation (METa) assembly for extremely efficient construction of functional metagenomic libraries
    Article Snippet: .. Following hybridization, NEBuilder HiFi assembly master mix polymerase and ligase fill gaps and ligate nicks, respectively, to produce a covalently sealed construct for transformation . .. Expression and purification of transposase enzyme and 1 kb to 10 kb DNA tagmentation We began our experiments with the goal of increasing DNA efficiency during metagenomic library preparation by adapting the tagmentation work flow used in shotgun sequencing library protocols ( , ).

    Article Title: Optimization of insect odorant receptor trafficking and functional expression via transient transfection in HEK293 cells
    Article Snippet: .. The mRho.V5.mER.hOr47a construct was inserted in the pDmelOR vector after linearization with EcoRI, using the BI-R.E.hOr47a_fwd and BI-R.E.hOr47a_rev using the NEBuilder HiFi DNA Assembly Master Mix (pDmelOR-mRho.V5.mER.hOr47a, or pDmelOR-R.E.hOr47a). .. Moreover, a human codon-optimized versions of D. melanogaster Or56a (hOr56a) was synthesized by Eurofins Genomics GmbH and inserted in the pDmelOR vector after linearization with EcoRI, together with the mRho.V5.mER tag using the following set of primers: hOr56a_fwd, hOr56a_rev, hOr56aTag_fwd, hOr56aTag_rev (pDmelOR-mRho.V5.mER.hOr56a, or pDmelOR-R.E.hOr56a).

    Protein Purification:

    Article Title: Disruption of ATRX-RNA interactions uncovers roles in ATRX localization and PRC2 function
    Article Snippet: .. Protein purification Full-length FLAG-ATRX-HA, FLAG-ATRXΔRBR-HA, and FLAG-ATRX helicase domain were generated using NEBuilder (NEB #E2621S). .. Proteins were purified from Sf9 cells using M2 agarose beads (Sigma #M8823-1ML).

    Generated:

    Article Title: Disruption of ATRX-RNA interactions uncovers roles in ATRX localization and PRC2 function
    Article Snippet: .. Protein purification Full-length FLAG-ATRX-HA, FLAG-ATRXΔRBR-HA, and FLAG-ATRX helicase domain were generated using NEBuilder (NEB #E2621S). .. Proteins were purified from Sf9 cells using M2 agarose beads (Sigma #M8823-1ML).

    Expressing:

    Article Title: User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome
    Article Snippet: .. Delete ORF1-ORF2 in pKFAV4 pKFAV4AP was digested with AgeI/NheI, and the fragment of 4942 bp was recovered; PCR was performed to amplify AgeI-ORF1 fragment (Open Reading Frame, ORF; from upstream of AgeI site in ORF0 to ORF1; 204 bp) with primers of 1707KFAV4AgeIF/R ( ) using pKFAV4AP as the template (Q5 High-Fidelity DNA Polymerase); PCR was performed to amplify GFP expression cassette (1672 bp) with primers of 1707F02GFPF/R using pAd5GFP as the template; and these three fragments were fused together to generate plasmid pKFAV4APN-GFP ( pKFAV4AP N heI -GFP ) by DNA assembly. pKFAV4APN-GFP was digested with NheI, treated with CIP (Calf Intestinal Alkaline Phosphatase; New England Biolabs), and ligated with the large fragment of NheI-digested pKFAV4AP (9840 bp) to generate plasmid pKFAV4AP-GFP (16,575 bp). pKFAV4AP-GFP was digested with PacI and fused to the large fragment of AvrII-digested pKFAV4 (29,468 bp) to generate pKFAV4-GFP (45,981 bp) by DNA assembly (NEBuilderHiFi DNA Assembly Master Mix). .. Replace Coding Sequence of FAdV-4 ORF4 with That of mCherry in pKFAV4-GFP PvuI and BamHI were single cutters flanking ORF4 coding sequence (CDS) in pKFAV4APN-GFP.

    Polymerase Chain Reaction:

    Article Title: User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome
    Article Snippet: .. Delete ORF1-ORF2 in pKFAV4 pKFAV4AP was digested with AgeI/NheI, and the fragment of 4942 bp was recovered; PCR was performed to amplify AgeI-ORF1 fragment (Open Reading Frame, ORF; from upstream of AgeI site in ORF0 to ORF1; 204 bp) with primers of 1707KFAV4AgeIF/R ( ) using pKFAV4AP as the template (Q5 High-Fidelity DNA Polymerase); PCR was performed to amplify GFP expression cassette (1672 bp) with primers of 1707F02GFPF/R using pAd5GFP as the template; and these three fragments were fused together to generate plasmid pKFAV4APN-GFP ( pKFAV4AP N heI -GFP ) by DNA assembly. pKFAV4APN-GFP was digested with NheI, treated with CIP (Calf Intestinal Alkaline Phosphatase; New England Biolabs), and ligated with the large fragment of NheI-digested pKFAV4AP (9840 bp) to generate plasmid pKFAV4AP-GFP (16,575 bp). pKFAV4AP-GFP was digested with PacI and fused to the large fragment of AvrII-digested pKFAV4 (29,468 bp) to generate pKFAV4-GFP (45,981 bp) by DNA assembly (NEBuilderHiFi DNA Assembly Master Mix). .. Replace Coding Sequence of FAdV-4 ORF4 with That of mCherry in pKFAV4-GFP PvuI and BamHI were single cutters flanking ORF4 coding sequence (CDS) in pKFAV4APN-GFP.

    Article Title: Antagonism of PP2A is an independent and conserved function of HIV-1 Vif and causes cell cycle arrest
    Article Snippet: .. The two PCR products were then assembled into the vector using the NEBuilder HiFi DNA Assembly Master Mix (NEB). .. Sequences for all primers used are tabulated in Supplementary file 1 (PCR primers for Vif mutant library construction).

    Transformation Assay:

    Article Title: Mosaic Ends Tagmentation (METa) assembly for extremely efficient construction of functional metagenomic libraries
    Article Snippet: .. Following hybridization, NEBuilder HiFi assembly master mix polymerase and ligase fill gaps and ligate nicks, respectively, to produce a covalently sealed construct for transformation . .. Expression and purification of transposase enzyme and 1 kb to 10 kb DNA tagmentation We began our experiments with the goal of increasing DNA efficiency during metagenomic library preparation by adapting the tagmentation work flow used in shotgun sequencing library protocols ( , ).

    Plasmid Preparation:

    Article Title: User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome
    Article Snippet: .. Delete ORF1-ORF2 in pKFAV4 pKFAV4AP was digested with AgeI/NheI, and the fragment of 4942 bp was recovered; PCR was performed to amplify AgeI-ORF1 fragment (Open Reading Frame, ORF; from upstream of AgeI site in ORF0 to ORF1; 204 bp) with primers of 1707KFAV4AgeIF/R ( ) using pKFAV4AP as the template (Q5 High-Fidelity DNA Polymerase); PCR was performed to amplify GFP expression cassette (1672 bp) with primers of 1707F02GFPF/R using pAd5GFP as the template; and these three fragments were fused together to generate plasmid pKFAV4APN-GFP ( pKFAV4AP N heI -GFP ) by DNA assembly. pKFAV4APN-GFP was digested with NheI, treated with CIP (Calf Intestinal Alkaline Phosphatase; New England Biolabs), and ligated with the large fragment of NheI-digested pKFAV4AP (9840 bp) to generate plasmid pKFAV4AP-GFP (16,575 bp). pKFAV4AP-GFP was digested with PacI and fused to the large fragment of AvrII-digested pKFAV4 (29,468 bp) to generate pKFAV4-GFP (45,981 bp) by DNA assembly (NEBuilderHiFi DNA Assembly Master Mix). .. Replace Coding Sequence of FAdV-4 ORF4 with That of mCherry in pKFAV4-GFP PvuI and BamHI were single cutters flanking ORF4 coding sequence (CDS) in pKFAV4APN-GFP.

    Article Title: Antagonism of PP2A is an independent and conserved function of HIV-1 Vif and causes cell cycle arrest
    Article Snippet: .. The two PCR products were then assembled into the vector using the NEBuilder HiFi DNA Assembly Master Mix (NEB). .. Sequences for all primers used are tabulated in Supplementary file 1 (PCR primers for Vif mutant library construction).

    Article Title: Mosaic Ends Tagmentation (METa) assembly for extremely efficient construction of functional metagenomic libraries
    Article Snippet: .. The resulting DNA would be mixed with the NEBuilder HiFi assembly master mix, resulting in 3’ overhangs able to hybridize to complementary sequences on linearized vector. .. Following hybridization, NEBuilder HiFi assembly master mix polymerase and ligase fill gaps and ligate nicks, respectively, to produce a covalently sealed construct for transformation .

    Article Title: Optimization of insect odorant receptor trafficking and functional expression via transient transfection in HEK293 cells
    Article Snippet: .. The mRho.V5.mER.hOr47a construct was inserted in the pDmelOR vector after linearization with EcoRI, using the BI-R.E.hOr47a_fwd and BI-R.E.hOr47a_rev using the NEBuilder HiFi DNA Assembly Master Mix (pDmelOR-mRho.V5.mER.hOr47a, or pDmelOR-R.E.hOr47a). .. Moreover, a human codon-optimized versions of D. melanogaster Or56a (hOr56a) was synthesized by Eurofins Genomics GmbH and inserted in the pDmelOR vector after linearization with EcoRI, together with the mRho.V5.mER tag using the following set of primers: hOr56a_fwd, hOr56a_rev, hOr56aTag_fwd, hOr56aTag_rev (pDmelOR-mRho.V5.mER.hOr56a, or pDmelOR-R.E.hOr56a).

    Hybridization:

    Article Title: Mosaic Ends Tagmentation (METa) assembly for extremely efficient construction of functional metagenomic libraries
    Article Snippet: .. Following hybridization, NEBuilder HiFi assembly master mix polymerase and ligase fill gaps and ligate nicks, respectively, to produce a covalently sealed construct for transformation . .. Expression and purification of transposase enzyme and 1 kb to 10 kb DNA tagmentation We began our experiments with the goal of increasing DNA efficiency during metagenomic library preparation by adapting the tagmentation work flow used in shotgun sequencing library protocols ( , ).

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    New England Biolabs nebuilder hifi assembly
    Colony PCR to determine library size and average insert length of mixed genome library prepared by METa assembly or blunt cloning Lanes 1, 25, 45, and 69: DNA ladders with top and brightest bands corresponding to 20 kb and 1.5 kb, respectively. Lanes 2-24, 26-44: Colonies from three replicate METa assemblies using <t>NEBuilder</t> <t>HiFi</t> (13 colonies per replicate, lanes 15, 30, and 44 from negative control sham colonies). Lanes 46-68, 70-88: Colonies from triplicate blunt ligation cloning reactions (13 colonies per replicate, lanes 59, 74, and 88 from negative control sham colonies). Reactions resulting in 500 bp amplicons indicate carriage of a no inserts vector. Reactions resulting in no visible band indicate technical failure of the colony PCR reaction.
    Nebuilder Hifi Assembly, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs flag atrxδrbr ha
    RBR-ID identifies regions within ATRX that bind RNA. a Top—Residue-level RBR-ID scores plotted along the primary sequence of human ATRX. Data are from eight biological replicates processed in two separate experiments. Bottom—Schematic for location of RNA binding peptides along human ATRX. PHD finger domain is in gray and helicase domain in dark blue. The region we designate the RBR is in green. b Coomassie gel of purified GST and GST-RBR (left), ATRX helicase domain (middle), and full-length ATRX and <t>ATRXΔRBR</t> (right) proteins. Representative image from three independent protein purifications is shown. c In vitro DNA/RNA IP with GST (gray) and GST-RBR (blue) proteins as indicated with MBP or Xist Repeat A DNA/RNA. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. d In vitro RNA IP with GST (left) and GST-RBR (right) proteins at indicated concentrations with MBP or Xist Repeat A RNA. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. e In vitro RNA IP with GST and GST-RBR and a mixture of MBP and Repeat A RNAs. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. f In vitro RNA IP with <t>FLAG-ATRX</t> helicase, GST-RBR and Repeat A RNA. Flag beads alone or GST are used as controls. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. g In vitro DNA/RNA IP with ATRX (gray) and ATRXΔRBR (blue) proteins as indicated with MBP or Xist Repeat A DNA/RNA. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. h RNA IP with purified proteins as indicated and total RNAs from MEFs. RNA is visualized by SYBR gold staining and immunoprecipitated proteins are detected by western blot. Source data are provided as a Source Data file. Representative image from three independent experiments is shown. Source data underlying Fig. 2b–h are provided as a Source Data file.
    Flag Atrxδrbr Ha, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Image Search Results


    Colony PCR to determine library size and average insert length of mixed genome library prepared by METa assembly or blunt cloning Lanes 1, 25, 45, and 69: DNA ladders with top and brightest bands corresponding to 20 kb and 1.5 kb, respectively. Lanes 2-24, 26-44: Colonies from three replicate METa assemblies using NEBuilder HiFi (13 colonies per replicate, lanes 15, 30, and 44 from negative control sham colonies). Lanes 46-68, 70-88: Colonies from triplicate blunt ligation cloning reactions (13 colonies per replicate, lanes 59, 74, and 88 from negative control sham colonies). Reactions resulting in 500 bp amplicons indicate carriage of a no inserts vector. Reactions resulting in no visible band indicate technical failure of the colony PCR reaction.

    Journal: bioRxiv

    Article Title: Mosaic Ends Tagmentation (METa) assembly for extremely efficient construction of functional metagenomic libraries

    doi: 10.1101/2021.02.01.429292

    Figure Lengend Snippet: Colony PCR to determine library size and average insert length of mixed genome library prepared by METa assembly or blunt cloning Lanes 1, 25, 45, and 69: DNA ladders with top and brightest bands corresponding to 20 kb and 1.5 kb, respectively. Lanes 2-24, 26-44: Colonies from three replicate METa assemblies using NEBuilder HiFi (13 colonies per replicate, lanes 15, 30, and 44 from negative control sham colonies). Lanes 46-68, 70-88: Colonies from triplicate blunt ligation cloning reactions (13 colonies per replicate, lanes 59, 74, and 88 from negative control sham colonies). Reactions resulting in 500 bp amplicons indicate carriage of a no inserts vector. Reactions resulting in no visible band indicate technical failure of the colony PCR reaction.

    Article Snippet: NEBuilder HiFi assembly was performed as before, with triplicate reactions containing 40 ng of inserts and 20 ng of pZE21-ME linear vector, and parallel triplicate sham reactions containing milliQ water in place of inserts.

    Techniques: Polymerase Chain Reaction, Clone Assay, Negative Control, Ligation, Plasmid Preparation

    Colony PCR to determine library size and average insert length of soil metagenomic DNA prepared by METa assembly (In-Fusion and NEBuilder HiFi) or blunt cloning Lanes 1, 17, 33, and 49: DNA ladders with brightest bands corresponding to 5 kb, 1.5 kb, and 500 bp. Lane 2: Single clone resulting from In-Fusion mediated METa assembly. Lanes 3-11, 12-21, and 22-31: Colonies from three replicate METa assemblies using NEBuilder HiFi. Lanes 32-41, 42-51, and 52-60: Colonies from triplicate blunt ligation cloning reactions. Lanes with bands at 500 bp correspond to amplification of vector backbone only (no inserts) while lanes with no band indicate colony PCR reaction failure

    Journal: bioRxiv

    Article Title: Mosaic Ends Tagmentation (METa) assembly for extremely efficient construction of functional metagenomic libraries

    doi: 10.1101/2021.02.01.429292

    Figure Lengend Snippet: Colony PCR to determine library size and average insert length of soil metagenomic DNA prepared by METa assembly (In-Fusion and NEBuilder HiFi) or blunt cloning Lanes 1, 17, 33, and 49: DNA ladders with brightest bands corresponding to 5 kb, 1.5 kb, and 500 bp. Lane 2: Single clone resulting from In-Fusion mediated METa assembly. Lanes 3-11, 12-21, and 22-31: Colonies from three replicate METa assemblies using NEBuilder HiFi. Lanes 32-41, 42-51, and 52-60: Colonies from triplicate blunt ligation cloning reactions. Lanes with bands at 500 bp correspond to amplification of vector backbone only (no inserts) while lanes with no band indicate colony PCR reaction failure

    Article Snippet: NEBuilder HiFi assembly was performed as before, with triplicate reactions containing 40 ng of inserts and 20 ng of pZE21-ME linear vector, and parallel triplicate sham reactions containing milliQ water in place of inserts.

    Techniques: Polymerase Chain Reaction, Clone Assay, Ligation, Amplification, Plasmid Preparation

    Blunt cloning protocol compared to METa assembly with NEBuilder HiFi or In-Fusion a) Transposase enzyme fragments DNA with 5’ mosaic end oligos. Inserts can be used as input for all three methods. All three protocols are compatible with linear pZE21-ME vector prepared by inverse PCR. b) Blunt cloning via end-repair and ligase. 5’ overhangs must be resolved by gap filling and phosphorylation using end-repair enzyme mixes. Blunt ended inserts can be ligated to blunt ended vector. c) METa assembly via In-Fusion enzyme mix. In-Fusion 3’ exonuclease activity is directly compatible with transposase fragments. Single stranded DNA overhangs on inserts and vector hybridize into a stable complex that can be transformed without filling gaps or covalently sealing nicks. d) METa assembly via NEBuilder HiFi enzyme mix. 5’ overhangs must be resolved by DNA polymerase gap filling. NEBuilder HiFi enzyme mix includes 5’ exonuclease to create 3’ overhangs which hybridize with target pZE21-ME. DNA polymerase fills in gaps and ligase seals nicks. e) pZE21-ME is prepared and linearized by inverse PCR and is compatible with all three DNA pipelines.

    Journal: bioRxiv

    Article Title: Mosaic Ends Tagmentation (METa) assembly for extremely efficient construction of functional metagenomic libraries

    doi: 10.1101/2021.02.01.429292

    Figure Lengend Snippet: Blunt cloning protocol compared to METa assembly with NEBuilder HiFi or In-Fusion a) Transposase enzyme fragments DNA with 5’ mosaic end oligos. Inserts can be used as input for all three methods. All three protocols are compatible with linear pZE21-ME vector prepared by inverse PCR. b) Blunt cloning via end-repair and ligase. 5’ overhangs must be resolved by gap filling and phosphorylation using end-repair enzyme mixes. Blunt ended inserts can be ligated to blunt ended vector. c) METa assembly via In-Fusion enzyme mix. In-Fusion 3’ exonuclease activity is directly compatible with transposase fragments. Single stranded DNA overhangs on inserts and vector hybridize into a stable complex that can be transformed without filling gaps or covalently sealing nicks. d) METa assembly via NEBuilder HiFi enzyme mix. 5’ overhangs must be resolved by DNA polymerase gap filling. NEBuilder HiFi enzyme mix includes 5’ exonuclease to create 3’ overhangs which hybridize with target pZE21-ME. DNA polymerase fills in gaps and ligase seals nicks. e) pZE21-ME is prepared and linearized by inverse PCR and is compatible with all three DNA pipelines.

    Article Snippet: NEBuilder HiFi assembly was performed as before, with triplicate reactions containing 40 ng of inserts and 20 ng of pZE21-ME linear vector, and parallel triplicate sham reactions containing milliQ water in place of inserts.

    Techniques: Clone Assay, Plasmid Preparation, Inverse PCR, Activity Assay, Transformation Assay

    Comparing metagenomic libraries prepared by assembly or blunt ligation Functional metagenomic libraries were created using METa assembly via In-Fusion assembly or NEBuilder HiFi assembly and compared to a library constructed using blunt ligation. All libraries used the same input DNA and were each prepared in triplicate. Error bars represent standard deviation of n=3 experiments. Comparisons between blunt ligation and NEBuilder HiFi METa assembly were made using unpaired two tailed t tests. Only a single In-Fusion colony was isolated and therefore we did not perform statistical tests on that method. a) Post-transformation culture titers normalized to the quantity of insert DNA used in the assembly or cloning reaction itself. b) Average insert size for plasmids containing an insert. Colony PCR was performed on the single In-Fusion colony, and 9 colonies were analyzed and averaged for each replicate NEBuilder HiFi assembly or blunt ligation reaction to give three data points per method. c) Final total library size or each replicate measured in gigabase pairs (Gb) of captured metagenomic DNA normalized to the amount of insert (μg) used during cloning or assembly.

    Journal: bioRxiv

    Article Title: Mosaic Ends Tagmentation (METa) assembly for extremely efficient construction of functional metagenomic libraries

    doi: 10.1101/2021.02.01.429292

    Figure Lengend Snippet: Comparing metagenomic libraries prepared by assembly or blunt ligation Functional metagenomic libraries were created using METa assembly via In-Fusion assembly or NEBuilder HiFi assembly and compared to a library constructed using blunt ligation. All libraries used the same input DNA and were each prepared in triplicate. Error bars represent standard deviation of n=3 experiments. Comparisons between blunt ligation and NEBuilder HiFi METa assembly were made using unpaired two tailed t tests. Only a single In-Fusion colony was isolated and therefore we did not perform statistical tests on that method. a) Post-transformation culture titers normalized to the quantity of insert DNA used in the assembly or cloning reaction itself. b) Average insert size for plasmids containing an insert. Colony PCR was performed on the single In-Fusion colony, and 9 colonies were analyzed and averaged for each replicate NEBuilder HiFi assembly or blunt ligation reaction to give three data points per method. c) Final total library size or each replicate measured in gigabase pairs (Gb) of captured metagenomic DNA normalized to the amount of insert (μg) used during cloning or assembly.

    Article Snippet: NEBuilder HiFi assembly was performed as before, with triplicate reactions containing 40 ng of inserts and 20 ng of pZE21-ME linear vector, and parallel triplicate sham reactions containing milliQ water in place of inserts.

    Techniques: Ligation, Functional Assay, Construct, Standard Deviation, Two Tailed Test, Isolation, Transformation Assay, Clone Assay, Polymerase Chain Reaction

    RBR-ID identifies regions within ATRX that bind RNA. a Top—Residue-level RBR-ID scores plotted along the primary sequence of human ATRX. Data are from eight biological replicates processed in two separate experiments. Bottom—Schematic for location of RNA binding peptides along human ATRX. PHD finger domain is in gray and helicase domain in dark blue. The region we designate the RBR is in green. b Coomassie gel of purified GST and GST-RBR (left), ATRX helicase domain (middle), and full-length ATRX and ATRXΔRBR (right) proteins. Representative image from three independent protein purifications is shown. c In vitro DNA/RNA IP with GST (gray) and GST-RBR (blue) proteins as indicated with MBP or Xist Repeat A DNA/RNA. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. d In vitro RNA IP with GST (left) and GST-RBR (right) proteins at indicated concentrations with MBP or Xist Repeat A RNA. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. e In vitro RNA IP with GST and GST-RBR and a mixture of MBP and Repeat A RNAs. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. f In vitro RNA IP with FLAG-ATRX helicase, GST-RBR and Repeat A RNA. Flag beads alone or GST are used as controls. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. g In vitro DNA/RNA IP with ATRX (gray) and ATRXΔRBR (blue) proteins as indicated with MBP or Xist Repeat A DNA/RNA. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. h RNA IP with purified proteins as indicated and total RNAs from MEFs. RNA is visualized by SYBR gold staining and immunoprecipitated proteins are detected by western blot. Source data are provided as a Source Data file. Representative image from three independent experiments is shown. Source data underlying Fig. 2b–h are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Disruption of ATRX-RNA interactions uncovers roles in ATRX localization and PRC2 function

    doi: 10.1038/s41467-020-15902-9

    Figure Lengend Snippet: RBR-ID identifies regions within ATRX that bind RNA. a Top—Residue-level RBR-ID scores plotted along the primary sequence of human ATRX. Data are from eight biological replicates processed in two separate experiments. Bottom—Schematic for location of RNA binding peptides along human ATRX. PHD finger domain is in gray and helicase domain in dark blue. The region we designate the RBR is in green. b Coomassie gel of purified GST and GST-RBR (left), ATRX helicase domain (middle), and full-length ATRX and ATRXΔRBR (right) proteins. Representative image from three independent protein purifications is shown. c In vitro DNA/RNA IP with GST (gray) and GST-RBR (blue) proteins as indicated with MBP or Xist Repeat A DNA/RNA. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. d In vitro RNA IP with GST (left) and GST-RBR (right) proteins at indicated concentrations with MBP or Xist Repeat A RNA. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. e In vitro RNA IP with GST and GST-RBR and a mixture of MBP and Repeat A RNAs. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. f In vitro RNA IP with FLAG-ATRX helicase, GST-RBR and Repeat A RNA. Flag beads alone or GST are used as controls. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. g In vitro DNA/RNA IP with ATRX (gray) and ATRXΔRBR (blue) proteins as indicated with MBP or Xist Repeat A DNA/RNA. Data are presented as mean values +/− SEM. P values are calculated using two-sided Student’s t test. h RNA IP with purified proteins as indicated and total RNAs from MEFs. RNA is visualized by SYBR gold staining and immunoprecipitated proteins are detected by western blot. Source data are provided as a Source Data file. Representative image from three independent experiments is shown. Source data underlying Fig. 2b–h are provided as a Source Data file.

    Article Snippet: Protein purification Full-length FLAG-ATRX-HA, FLAG-ATRXΔRBR-HA, and FLAG-ATRX helicase domain were generated using NEBuilder (NEB #E2621S).

    Techniques: Sequencing, RNA Binding Assay, Purification, In Vitro, Staining, Immunoprecipitation, Western Blot