input dna  (New England Biolabs)


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    Name:
    NEBNext Single Cell Low Input cDNA Synthesis Amplification Module
    Description:
    NEBNext Single Cell Low Input cDNA Synthesis Amplification Module 96 rxns
    Catalog Number:
    e6421l
    Price:
    2210
    Size:
    96 rxns
    Category:
    DNA Template Preparation for PCR
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    Structured Review

    New England Biolabs input dna
    NEBNext Single Cell Low Input cDNA Synthesis Amplification Module
    NEBNext Single Cell Low Input cDNA Synthesis Amplification Module 96 rxns
    https://www.bioz.com/result/input dna/product/New England Biolabs
    Average 94 stars, based on 106 article reviews
    Price from $9.99 to $1999.99
    input dna - by Bioz Stars, 2020-07
    94/100 stars

    Images

    1) Product Images from "GATA-1-dependent histone H3K27ac mediates erythroid cell-specific interaction between CTCF sites"

    Article Title: GATA-1-dependent histone H3K27ac mediates erythroid cell-specific interaction between CTCF sites

    Journal: bioRxiv

    doi: 10.1101/2020.05.14.095547

    Chromatin structures at CTCF sites around the β-globin locus having deletion of GATA-1 binding motifs. ChIP was performed with antibodies specific to CTCF (A), Rad21 (B), histone H3 (C) and H3K27ac (D) in control cells (Con) and HS3ΔΔGA and HS2ΔGA clones. Amounts of immunoprecipitated DNA were determined as described in Fig 1C . The mouse Necdin gene served as an internal control. The results are the means of four independent experiments ± SEM. *P
    Figure Legend Snippet: Chromatin structures at CTCF sites around the β-globin locus having deletion of GATA-1 binding motifs. ChIP was performed with antibodies specific to CTCF (A), Rad21 (B), histone H3 (C) and H3K27ac (D) in control cells (Con) and HS3ΔΔGA and HS2ΔGA clones. Amounts of immunoprecipitated DNA were determined as described in Fig 1C . The mouse Necdin gene served as an internal control. The results are the means of four independent experiments ± SEM. *P

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Clone Assay, Immunoprecipitation

    Chromatin interaction between CTCF sites around the β-globin locus in TSA treated MEL/ch11 cells. (A) MEL/ch11 cells were treated with 25 ng/ml of TSA for 6 h or 24 h. Histone H3 acetylated at K27 was detected by Western blotting in nuclear extract from control cells (Con) and cells treated with TSA. Histone H3 was used as an experimental control. (B) H3K27ac was determined in CTCF sites around the β-globin locus by ChIP. DNA immunoprecipitated by H3K27ac antibodies were quantitatively compared with DNA immunoprecipitated by H3, and then normalized to value in control cells. Normal IgG (IgG) was used as an experimental negative control. (C) Relative cross-linking frequencies was determined between CTCF sites around the β-globin locus in 3C assay as described in Fig 1E . Fragments containing C5 and C3 were used as anchors. Occupancies of CTCF (D) and Rad21 (E) were determined at the CTCF sites by ChIP. Results are presented as the means ± SEM of four to six independent experiments in ChIP and 3C assay. *P
    Figure Legend Snippet: Chromatin interaction between CTCF sites around the β-globin locus in TSA treated MEL/ch11 cells. (A) MEL/ch11 cells were treated with 25 ng/ml of TSA for 6 h or 24 h. Histone H3 acetylated at K27 was detected by Western blotting in nuclear extract from control cells (Con) and cells treated with TSA. Histone H3 was used as an experimental control. (B) H3K27ac was determined in CTCF sites around the β-globin locus by ChIP. DNA immunoprecipitated by H3K27ac antibodies were quantitatively compared with DNA immunoprecipitated by H3, and then normalized to value in control cells. Normal IgG (IgG) was used as an experimental negative control. (C) Relative cross-linking frequencies was determined between CTCF sites around the β-globin locus in 3C assay as described in Fig 1E . Fragments containing C5 and C3 were used as anchors. Occupancies of CTCF (D) and Rad21 (E) were determined at the CTCF sites by ChIP. Results are presented as the means ± SEM of four to six independent experiments in ChIP and 3C assay. *P

    Techniques Used: Western Blot, Chromatin Immunoprecipitation, Immunoprecipitation, Negative Control

    2) Product Images from "DNA-binding domain fusions enhance the targeting range and precision of Cas9"

    Article Title: DNA-binding domain fusions enhance the targeting range and precision of Cas9

    Journal: Nature methods

    doi: 10.1038/nmeth.3624

    Attenuating nuclease activity of SpCas9. ( a ) Four PAM-interacting amino acids neighboring the nGG PAM (magenta) in the structure of SpCas9 7 . Arginines at positions 1333 and 1335 were mutated to attenuate DNA-binding affinity of SpCas9. ( b ) Activity profile of SpCas9 (blue) or SpCas9-Zif268 (red) bearing lysine or serine substitutions at positions 1333 or 1335 in the PAM interaction domain in comparison to wild-type (WT) SpCas9. Reporter assays were performed in HEK293T cells. Bar heights represent means from three independent biological replicates performed on different days. Error bars indicate standard error of the mean. ( c ) T7 Endonuclease I (T7EI) assays on PCR products spanning a genomic target site (underlined) with an NGG PAM (magenta) and neighboring Zif268 site (orange) for SpCas9 or SpCas9 mutants with or without a Zif268 fusion. For SpCas9 MT2 SpCas9 MT3 , robust nuclease activity is only observed when Zif268 is fused to the C-terminus. The gel image is representative of T7EI assays at this genomic target site, where cleaved products are noted by magenta arrowheads. ( d ) Quantification of average T7EI-based lesion rates at the PLXNB2 locus from three independent biological replicates performed on different days in HEK293T cells ( Supplementary Fig. 7 ). Error bars indicate standard error of the mean.
    Figure Legend Snippet: Attenuating nuclease activity of SpCas9. ( a ) Four PAM-interacting amino acids neighboring the nGG PAM (magenta) in the structure of SpCas9 7 . Arginines at positions 1333 and 1335 were mutated to attenuate DNA-binding affinity of SpCas9. ( b ) Activity profile of SpCas9 (blue) or SpCas9-Zif268 (red) bearing lysine or serine substitutions at positions 1333 or 1335 in the PAM interaction domain in comparison to wild-type (WT) SpCas9. Reporter assays were performed in HEK293T cells. Bar heights represent means from three independent biological replicates performed on different days. Error bars indicate standard error of the mean. ( c ) T7 Endonuclease I (T7EI) assays on PCR products spanning a genomic target site (underlined) with an NGG PAM (magenta) and neighboring Zif268 site (orange) for SpCas9 or SpCas9 mutants with or without a Zif268 fusion. For SpCas9 MT2 SpCas9 MT3 , robust nuclease activity is only observed when Zif268 is fused to the C-terminus. The gel image is representative of T7EI assays at this genomic target site, where cleaved products are noted by magenta arrowheads. ( d ) Quantification of average T7EI-based lesion rates at the PLXNB2 locus from three independent biological replicates performed on different days in HEK293T cells ( Supplementary Fig. 7 ). Error bars indicate standard error of the mean.

    Techniques Used: Activity Assay, Binding Assay, Polymerase Chain Reaction

    3) Product Images from "Facile, High Quality Sequencing of Bacterial Genomes from Small Amounts of DNA"

    Article Title: Facile, High Quality Sequencing of Bacterial Genomes from Small Amounts of DNA

    Journal: International Journal of Genomics

    doi: 10.1155/2014/434575

    Evenness of coverage for B. anthracis (a) and E. coli (b) genome sequencing using NEBNext library preparation with 100 ng DNA input (top graph) and TruSeq library preparation with 1,000 ng DNA input (bottom graph). Plots are normalized by the average coverage in each figure.
    Figure Legend Snippet: Evenness of coverage for B. anthracis (a) and E. coli (b) genome sequencing using NEBNext library preparation with 100 ng DNA input (top graph) and TruSeq library preparation with 1,000 ng DNA input (bottom graph). Plots are normalized by the average coverage in each figure.

    Techniques Used: Sequencing

    Evenness of coverage plots for B. thailandensis A ((a) is chromosome 1 and (b) is chromosome 2) and B. thailandensis B ((c) is chromosome 1 and (d) is chromosome 2) genome sequencing. Top graph within each panel shows NEBNext library preparation data with 10 ng or 100 ng DNA input (black color is for 10 ng samples and red color is for 100 ng samples). Bottom graph within each panel shows TruSeq library preparation data with 1,000 ng DNA input. Plots are normalized by the average coverage in each figure.
    Figure Legend Snippet: Evenness of coverage plots for B. thailandensis A ((a) is chromosome 1 and (b) is chromosome 2) and B. thailandensis B ((c) is chromosome 1 and (d) is chromosome 2) genome sequencing. Top graph within each panel shows NEBNext library preparation data with 10 ng or 100 ng DNA input (black color is for 10 ng samples and red color is for 100 ng samples). Bottom graph within each panel shows TruSeq library preparation data with 1,000 ng DNA input. Plots are normalized by the average coverage in each figure.

    Techniques Used: Sequencing

    4) Product Images from "DNA-binding domain fusions enhance the targeting range and precision of Cas9"

    Article Title: DNA-binding domain fusions enhance the targeting range and precision of Cas9

    Journal: Nature methods

    doi: 10.1038/nmeth.3624

    Attenuating nuclease activity of SpCas9. ( a ) Four PAM-interacting amino acids neighboring the nGG PAM (magenta) in the structure of SpCas9 7 . Arginines at positions 1333 and 1335 were mutated to attenuate DNA-binding affinity of SpCas9. ( b ) Activity profile of SpCas9 (blue) or SpCas9-Zif268 (red) bearing lysine or serine substitutions at positions 1333 or 1335 in the PAM interaction domain in comparison to wild-type (WT) SpCas9. Reporter assays were performed in HEK293T cells. Bar heights represent means from three independent biological replicates performed on different days. Error bars indicate standard error of the mean. ( c ) T7 Endonuclease I (T7EI) assays on PCR products spanning a genomic target site (underlined) with an NGG PAM (magenta) and neighboring Zif268 site (orange) for SpCas9 or SpCas9 mutants with or without a Zif268 fusion. For SpCas9 MT2 SpCas9 MT3 , robust nuclease activity is only observed when Zif268 is fused to the C-terminus. The gel image is representative of T7EI assays at this genomic target site, where cleaved products are noted by magenta arrowheads. ( d ) Quantification of average T7EI-based lesion rates at the PLXNB2 locus from three independent biological replicates performed on different days in HEK293T cells ( Supplementary Fig. 7 ). Error bars indicate standard error of the mean.
    Figure Legend Snippet: Attenuating nuclease activity of SpCas9. ( a ) Four PAM-interacting amino acids neighboring the nGG PAM (magenta) in the structure of SpCas9 7 . Arginines at positions 1333 and 1335 were mutated to attenuate DNA-binding affinity of SpCas9. ( b ) Activity profile of SpCas9 (blue) or SpCas9-Zif268 (red) bearing lysine or serine substitutions at positions 1333 or 1335 in the PAM interaction domain in comparison to wild-type (WT) SpCas9. Reporter assays were performed in HEK293T cells. Bar heights represent means from three independent biological replicates performed on different days. Error bars indicate standard error of the mean. ( c ) T7 Endonuclease I (T7EI) assays on PCR products spanning a genomic target site (underlined) with an NGG PAM (magenta) and neighboring Zif268 site (orange) for SpCas9 or SpCas9 mutants with or without a Zif268 fusion. For SpCas9 MT2 SpCas9 MT3 , robust nuclease activity is only observed when Zif268 is fused to the C-terminus. The gel image is representative of T7EI assays at this genomic target site, where cleaved products are noted by magenta arrowheads. ( d ) Quantification of average T7EI-based lesion rates at the PLXNB2 locus from three independent biological replicates performed on different days in HEK293T cells ( Supplementary Fig. 7 ). Error bars indicate standard error of the mean.

    Techniques Used: Activity Assay, Binding Assay, Polymerase Chain Reaction

    5) Product Images from "DNA-binding domain fusions enhance the targeting range and precision of Cas9"

    Article Title: DNA-binding domain fusions enhance the targeting range and precision of Cas9

    Journal: Nature methods

    doi: 10.1038/nmeth.3624

    Attenuating nuclease activity of SpCas9. ( a ) Four PAM-interacting amino acids neighboring the nGG PAM (magenta) in the structure of SpCas9 7 . Arginines at positions 1333 and 1335 were mutated to attenuate DNA-binding affinity of SpCas9. ( b ) Activity profile of SpCas9 (blue) or SpCas9-Zif268 (red) bearing lysine or serine substitutions at positions 1333 or 1335 in the PAM interaction domain in comparison to wild-type (WT) SpCas9. Reporter assays were performed in HEK293T cells. Bar heights represent means from three independent biological replicates performed on different days. Error bars indicate standard error of the mean. ( c ) T7 Endonuclease I (T7EI) assays on PCR products spanning a genomic target site (underlined) with an NGG PAM (magenta) and neighboring Zif268 site (orange) for SpCas9 or SpCas9 mutants with or without a Zif268 fusion. For SpCas9 MT2 SpCas9 MT3 , robust nuclease activity is only observed when Zif268 is fused to the C-terminus. The gel image is representative of T7EI assays at this genomic target site, where cleaved products are noted by magenta arrowheads. ( d ) Quantification of average T7EI-based lesion rates at the PLXNB2 locus from three independent biological replicates performed on different days in HEK293T cells ( Supplementary Fig. 7 ). Error bars indicate standard error of the mean.
    Figure Legend Snippet: Attenuating nuclease activity of SpCas9. ( a ) Four PAM-interacting amino acids neighboring the nGG PAM (magenta) in the structure of SpCas9 7 . Arginines at positions 1333 and 1335 were mutated to attenuate DNA-binding affinity of SpCas9. ( b ) Activity profile of SpCas9 (blue) or SpCas9-Zif268 (red) bearing lysine or serine substitutions at positions 1333 or 1335 in the PAM interaction domain in comparison to wild-type (WT) SpCas9. Reporter assays were performed in HEK293T cells. Bar heights represent means from three independent biological replicates performed on different days. Error bars indicate standard error of the mean. ( c ) T7 Endonuclease I (T7EI) assays on PCR products spanning a genomic target site (underlined) with an NGG PAM (magenta) and neighboring Zif268 site (orange) for SpCas9 or SpCas9 mutants with or without a Zif268 fusion. For SpCas9 MT2 SpCas9 MT3 , robust nuclease activity is only observed when Zif268 is fused to the C-terminus. The gel image is representative of T7EI assays at this genomic target site, where cleaved products are noted by magenta arrowheads. ( d ) Quantification of average T7EI-based lesion rates at the PLXNB2 locus from three independent biological replicates performed on different days in HEK293T cells ( Supplementary Fig. 7 ). Error bars indicate standard error of the mean.

    Techniques Used: Activity Assay, Binding Assay, Polymerase Chain Reaction

    6) Product Images from "The DNA methylation landscape of human melanoma"

    Article Title: The DNA methylation landscape of human melanoma

    Journal: Genomics

    doi: 10.1016/j.ygeno.2015.09.004

    2.1. MIRA-seq identifies numerous tumor-specific DNA methylation peaks and potential DNA methylation markers for melanoma
    Figure Legend Snippet: 2.1. MIRA-seq identifies numerous tumor-specific DNA methylation peaks and potential DNA methylation markers for melanoma

    Techniques Used: DNA Methylation Assay

    7) Product Images from "STAT3 acts through pre-existing nucleosome-depleted regions bound by FOS during an epigenetic switch linking inflammation to cancer"

    Article Title: STAT3 acts through pre-existing nucleosome-depleted regions bound by FOS during an epigenetic switch linking inflammation to cancer

    Journal: Epigenetics & Chromatin

    doi: 10.1186/1756-8935-8-7

    NF-κB binds a subset of FAIRE sites independent of STAT3. (A) Transcription factor binding motifs enriched within FAIRE sites that were ±500 kb from the TSS of the genes in each indicated group. STAT3-dependent gene groups are separated based on whether their expression increases or decreases in response to siSTAT3. (B) Occupancy of NF-κB DNA binding site motifs, as a function of increasing motif quality score, within FAIRE-seq regions and in non-FAIRE regions. (C) Correlation matrix of the ChIP-seq signal at FAIRE sites. Read counts for each of the factors and conditions measured by ChIP-seq, normalized to input, were used to calculate the correlation matrix. The resulting correlation matrix is symmetric (same datasets represented on rows and columns) which was clustered by row and column to order datasets based on the similarity of binding within FAIRE sites. The correlation matrix indicates that NF-κB correlates well with itself but not to other factors, indicating that NF-κB binds an independent subset of sites. FOS and STAT3 however occupy the same FAIRE sites more frequently than other factors.
    Figure Legend Snippet: NF-κB binds a subset of FAIRE sites independent of STAT3. (A) Transcription factor binding motifs enriched within FAIRE sites that were ±500 kb from the TSS of the genes in each indicated group. STAT3-dependent gene groups are separated based on whether their expression increases or decreases in response to siSTAT3. (B) Occupancy of NF-κB DNA binding site motifs, as a function of increasing motif quality score, within FAIRE-seq regions and in non-FAIRE regions. (C) Correlation matrix of the ChIP-seq signal at FAIRE sites. Read counts for each of the factors and conditions measured by ChIP-seq, normalized to input, were used to calculate the correlation matrix. The resulting correlation matrix is symmetric (same datasets represented on rows and columns) which was clustered by row and column to order datasets based on the similarity of binding within FAIRE sites. The correlation matrix indicates that NF-κB correlates well with itself but not to other factors, indicating that NF-κB binds an independent subset of sites. FOS and STAT3 however occupy the same FAIRE sites more frequently than other factors.

    Techniques Used: Binding Assay, Expressing, Chromatin Immunoprecipitation

    STAT3 binding profiles before and during transformation. (A) Western blots of protein extracts from TAM-treated MCF10A-ER-Src cells. (B) Distribution of STAT3 occupancy at RefSeq gene features. “All STAT3” represents all treatments/time points, i.e., cumulative. “Differential” refers to STAT3 occupancy in transformed cells only. (C) Occupancy of STAT3 DNA binding site motifs by STAT3 protein, as a function of increasing motif quality score, within FAIRE-seq regions and in non-FAIRE regions. Data was from all STAT3 conditions/time points. (D) Gene ontology terms associated with transformation differential STAT3 ChIP-seq sites and the overlap between at 4, 12, and 36 h post TAM treatment.
    Figure Legend Snippet: STAT3 binding profiles before and during transformation. (A) Western blots of protein extracts from TAM-treated MCF10A-ER-Src cells. (B) Distribution of STAT3 occupancy at RefSeq gene features. “All STAT3” represents all treatments/time points, i.e., cumulative. “Differential” refers to STAT3 occupancy in transformed cells only. (C) Occupancy of STAT3 DNA binding site motifs by STAT3 protein, as a function of increasing motif quality score, within FAIRE-seq regions and in non-FAIRE regions. Data was from all STAT3 conditions/time points. (D) Gene ontology terms associated with transformation differential STAT3 ChIP-seq sites and the overlap between at 4, 12, and 36 h post TAM treatment.

    Techniques Used: Binding Assay, Transformation Assay, Western Blot, Chromatin Immunoprecipitation

    Cooperation of STAT3 and FOS sites during transformation. (A) AP-1 factors during transformation and their transcriptional dependence on STAT3. Shown are the normalized RNA expression microarray levels at 4 and 24 h post EtOH or TAM treatment in samples transfected with siSCM (scrambled control) or siSTAT3. (B) Occupancy of FOS DNA binding site motifs, as a function of increasing motif quality score, within FAIRE-seq regions and in non-FAIRE regions. (C) All FOS and STAT3 sites from each time point that directly overlap or overlap only at FAIRE sites. (D) Transformation-dependent differential STAT3 sites directly overlapping all FOS sites from 4, 12, and 24 h post induction.
    Figure Legend Snippet: Cooperation of STAT3 and FOS sites during transformation. (A) AP-1 factors during transformation and their transcriptional dependence on STAT3. Shown are the normalized RNA expression microarray levels at 4 and 24 h post EtOH or TAM treatment in samples transfected with siSCM (scrambled control) or siSTAT3. (B) Occupancy of FOS DNA binding site motifs, as a function of increasing motif quality score, within FAIRE-seq regions and in non-FAIRE regions. (C) All FOS and STAT3 sites from each time point that directly overlap or overlap only at FAIRE sites. (D) Transformation-dependent differential STAT3 sites directly overlapping all FOS sites from 4, 12, and 24 h post induction.

    Techniques Used: Transformation Assay, RNA Expression, Microarray, Transfection, Binding Assay

    8) Product Images from "MIRA-seq for DNA methylation analysis of CpG islands"

    Article Title: MIRA-seq for DNA methylation analysis of CpG islands

    Journal: Epigenomics

    doi: 10.2217/epi.15.33

    Example of MIRA-seq profiles used to identify cancer-associated DNA hypermethylation Data are for two samples of normal melanocytes (N, green) and four melanoma tumors (T, blue). The data are displayed in the Integrative Genomics Viewer and an area of chromosome 19 is shown. The vertical arrow points to the gene CCDC8 , which becomes methylated in the gene body in melanoma.
    Figure Legend Snippet: Example of MIRA-seq profiles used to identify cancer-associated DNA hypermethylation Data are for two samples of normal melanocytes (N, green) and four melanoma tumors (T, blue). The data are displayed in the Integrative Genomics Viewer and an area of chromosome 19 is shown. The vertical arrow points to the gene CCDC8 , which becomes methylated in the gene body in melanoma.

    Techniques Used: Methylation

    Validation of MIRA-seq peaks by sodium bisulfite sequencing (A) MIRA-seq was performed on DNA from normal melanocytes (N, green) and three melanoma samples (T1, T2, T3, blue). The vertical arrow indicates the region of the HOXA9 gene analyzed by bisulfite sequencing. (B) Bisulfite sequence data for normal melanocytes. (C–E) Bisulfite sequence data for three melanoma tumors. Open circles: unmethylated CpG sequences; black circles: methylated CpG sequences. The percentages indicate percentage of methylated CpGs.
    Figure Legend Snippet: Validation of MIRA-seq peaks by sodium bisulfite sequencing (A) MIRA-seq was performed on DNA from normal melanocytes (N, green) and three melanoma samples (T1, T2, T3, blue). The vertical arrow indicates the region of the HOXA9 gene analyzed by bisulfite sequencing. (B) Bisulfite sequence data for normal melanocytes. (C–E) Bisulfite sequence data for three melanoma tumors. Open circles: unmethylated CpG sequences; black circles: methylated CpG sequences. The percentages indicate percentage of methylated CpGs.

    Techniques Used: Methylation Sequencing, Sequencing, Methylation

    9) Product Images from "The DNA methylation landscape of human melanoma"

    Article Title: The DNA methylation landscape of human melanoma

    Journal: Genomics

    doi: 10.1016/j.ygeno.2015.09.004

    2.1. MIRA-seq identifies numerous tumor-specific DNA methylation peaks and potential DNA methylation markers for melanoma
    Figure Legend Snippet: 2.1. MIRA-seq identifies numerous tumor-specific DNA methylation peaks and potential DNA methylation markers for melanoma

    Techniques Used: DNA Methylation Assay

    10) Product Images from "Early growth response protein-1 mediates lipotoxicity-associated placental inflammation: role in maternal obesity"

    Article Title: Early growth response protein-1 mediates lipotoxicity-associated placental inflammation: role in maternal obesity

    Journal: American Journal of Physiology - Endocrinology and Metabolism

    doi: 10.1152/ajpendo.00076.2013

    Transcriptional regulation of early growth response protein-1 (EGR-1) in response to PA or PA + TNFα. A: EGR1 mRNA expression following exposure to PA or PA + TNFα over 24 h. Values were normalized to cyclophillin mRNA ( n = 6/group). B : GO term (biological processes) enrichment of genes with differential H3K4 trimethylation (me3) following PA treatment (500 μM, 24 h). Genome-wide H3K4me3 was assayed using chromatin immunoprecipitation (ChIP)-seq. C : alignment of H3K4me3 reads following ChIP-seq showing greater read density at the EGR1 gene locus following PA treatment. From top , tracks showing gene, mRNA, and coding DNA sequence (CDS) are in red, followed by control and PA groups. Read densities are shown as a heat map (red being high, blue being low) showing greater H3K4me3 in the PA group. D : a schematic of the human EGR1 promoter upstream of the transcriptional start site (+1). Response elements from SRF (SRE; white box), CREB (CRE; white oval), SP1 (black oval), Ets-1 (EBS; black box), and AP-1 (◇) are pictured in approximate location within the EGR1 promoter (not to scale). Arrows indicate PCR primer locations, and the dotted lines show areas of promoter amplification. E : ChIP was performed with ATF3, c-Jun, and SRF (or IgG as control) at 6 h following treatments with PA or PA + TNFα. PCR was performed using immunoprecipitated DNA or input DNA for various regions of the EGR1 promoter. Band intensity was quantified by densitometry and normalized to input DNA. Values are expressed relative to the control group. Statistical differences were determined using Student's t -test. *Significance, P ≤ 0.05. IP, immunoprecipitation.
    Figure Legend Snippet: Transcriptional regulation of early growth response protein-1 (EGR-1) in response to PA or PA + TNFα. A: EGR1 mRNA expression following exposure to PA or PA + TNFα over 24 h. Values were normalized to cyclophillin mRNA ( n = 6/group). B : GO term (biological processes) enrichment of genes with differential H3K4 trimethylation (me3) following PA treatment (500 μM, 24 h). Genome-wide H3K4me3 was assayed using chromatin immunoprecipitation (ChIP)-seq. C : alignment of H3K4me3 reads following ChIP-seq showing greater read density at the EGR1 gene locus following PA treatment. From top , tracks showing gene, mRNA, and coding DNA sequence (CDS) are in red, followed by control and PA groups. Read densities are shown as a heat map (red being high, blue being low) showing greater H3K4me3 in the PA group. D : a schematic of the human EGR1 promoter upstream of the transcriptional start site (+1). Response elements from SRF (SRE; white box), CREB (CRE; white oval), SP1 (black oval), Ets-1 (EBS; black box), and AP-1 (◇) are pictured in approximate location within the EGR1 promoter (not to scale). Arrows indicate PCR primer locations, and the dotted lines show areas of promoter amplification. E : ChIP was performed with ATF3, c-Jun, and SRF (or IgG as control) at 6 h following treatments with PA or PA + TNFα. PCR was performed using immunoprecipitated DNA or input DNA for various regions of the EGR1 promoter. Band intensity was quantified by densitometry and normalized to input DNA. Values are expressed relative to the control group. Statistical differences were determined using Student's t -test. *Significance, P ≤ 0.05. IP, immunoprecipitation.

    Techniques Used: Expressing, Genome Wide, Chromatin Immunoprecipitation, Sequencing, Polymerase Chain Reaction, Amplification, Immunoprecipitation

    11) Product Images from "Facile, High Quality Sequencing of Bacterial Genomes from Small Amounts of DNA"

    Article Title: Facile, High Quality Sequencing of Bacterial Genomes from Small Amounts of DNA

    Journal: International Journal of Genomics

    doi: 10.1155/2014/434575

    Evenness of coverage for B. anthracis (a) and E. coli (b) genome sequencing using NEBNext library preparation with 100 ng DNA input (top graph) and TruSeq library preparation with 1,000 ng DNA input (bottom graph). Plots are normalized by the average coverage in each figure.
    Figure Legend Snippet: Evenness of coverage for B. anthracis (a) and E. coli (b) genome sequencing using NEBNext library preparation with 100 ng DNA input (top graph) and TruSeq library preparation with 1,000 ng DNA input (bottom graph). Plots are normalized by the average coverage in each figure.

    Techniques Used: Sequencing

    Evenness of coverage plots for B. thailandensis A ((a) is chromosome 1 and (b) is chromosome 2) and B. thailandensis B ((c) is chromosome 1 and (d) is chromosome 2) genome sequencing. Top graph within each panel shows NEBNext library preparation data with 10 ng or 100 ng DNA input (black color is for 10 ng samples and red color is for 100 ng samples). Bottom graph within each panel shows TruSeq library preparation data with 1,000 ng DNA input. Plots are normalized by the average coverage in each figure.
    Figure Legend Snippet: Evenness of coverage plots for B. thailandensis A ((a) is chromosome 1 and (b) is chromosome 2) and B. thailandensis B ((c) is chromosome 1 and (d) is chromosome 2) genome sequencing. Top graph within each panel shows NEBNext library preparation data with 10 ng or 100 ng DNA input (black color is for 10 ng samples and red color is for 100 ng samples). Bottom graph within each panel shows TruSeq library preparation data with 1,000 ng DNA input. Plots are normalized by the average coverage in each figure.

    Techniques Used: Sequencing

    Related Articles

    cDNA Library Assay:

    Article Title: RNase H-dependent PCR-enabled T Cell Receptor sequencing (rhTCRseq) for Highly Specific and Efficient Targeted Sequencing of T Cell Receptor mRNA for Single-Cell and Repertoire Analysis
    Article Snippet: .. E6421L for cDNA library construction and E7805L for sequencing library construction can be purchased together as the NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina (New England BioLabs, cat. no. E6420L) .. Twin.tec PCR plate (384 wells, LoBind, skirted, PCR clean, colorless, Eppendorf, cat. no. 0030129547) Twin.tec PCR plate (96 wells, LoBind, skirted, PCR clean, colorless, Eppendorf, cat. no. 0030129512) 0.2-mL PCR 8-tube FLEX-FREE strip (attached clear flat caps, natural, USA Scientific, cat. no. 1402–4700) 20-μL 8-channel pipette (Pipet-Lite Multi Pipette L8–20XLS+, Rainin, cat. no. 17013803) 200-μL 8-channel pipette (Pipet-Lite Multi Pipette L8–200XLS+, Rainin, cat. no. 17013805) 2-μL pipette (Pipet-Lite LTS Pipette L-2XLS+, Rainin, cat. no. 17014393) 20-μL pipette (Pipet-Lite LTS Pipette L-20XLS+, Rainin, cat. no. 17014392) 200-μL pipette (Pipet-Lite LTS Pipette L-200XLS+, Rainin, cat. no. 17014391) 1000-μL pipette (Pipet-Lite LTS Pipette L-1000XLS+, Rainin, cat. no. 17014382) 20-μL pipette tips (RT-LTS-A-10μL-/F/L-960/10, Rainin, cat. no. 30389226) 200-μL pipette tips (RT-LTS-A-200μL-/F/L-960/10, Rainin, cat. no. 30389240) 1000-μL pipette tips (RT-LTS-A-1000μL-/F/L-768/8, Rainin, cat. no. 30389213) MagnaBot 384 magnetic separation stand (Promega, cat. no. V8241) 10x magnetic separation stand for 8-tube strip (10x Genomics, cat. no. 230003) DynaMag-96 side skirted magnetic separation stand (Thermo Fisher, cat. no. 12027) MagneSphere magnetic separation stand (12-hole, 1.5 mL vial, Promega, cat. no. Z5342) CoolRack XT cooling block for 384-well PCR plate (Corning, cat. no. 432055) for keeping plate cool on the deck of the Mantis liquid dispenser.

    Sequencing:

    Article Title: RNase H-dependent PCR-enabled T Cell Receptor sequencing (rhTCRseq) for Highly Specific and Efficient Targeted Sequencing of T Cell Receptor mRNA for Single-Cell and Repertoire Analysis
    Article Snippet: .. E6421L for cDNA library construction and E7805L for sequencing library construction can be purchased together as the NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina (New England BioLabs, cat. no. E6420L) .. Twin.tec PCR plate (384 wells, LoBind, skirted, PCR clean, colorless, Eppendorf, cat. no. 0030129547) Twin.tec PCR plate (96 wells, LoBind, skirted, PCR clean, colorless, Eppendorf, cat. no. 0030129512) 0.2-mL PCR 8-tube FLEX-FREE strip (attached clear flat caps, natural, USA Scientific, cat. no. 1402–4700) 20-μL 8-channel pipette (Pipet-Lite Multi Pipette L8–20XLS+, Rainin, cat. no. 17013803) 200-μL 8-channel pipette (Pipet-Lite Multi Pipette L8–200XLS+, Rainin, cat. no. 17013805) 2-μL pipette (Pipet-Lite LTS Pipette L-2XLS+, Rainin, cat. no. 17014393) 20-μL pipette (Pipet-Lite LTS Pipette L-20XLS+, Rainin, cat. no. 17014392) 200-μL pipette (Pipet-Lite LTS Pipette L-200XLS+, Rainin, cat. no. 17014391) 1000-μL pipette (Pipet-Lite LTS Pipette L-1000XLS+, Rainin, cat. no. 17014382) 20-μL pipette tips (RT-LTS-A-10μL-/F/L-960/10, Rainin, cat. no. 30389226) 200-μL pipette tips (RT-LTS-A-200μL-/F/L-960/10, Rainin, cat. no. 30389240) 1000-μL pipette tips (RT-LTS-A-1000μL-/F/L-768/8, Rainin, cat. no. 30389213) MagnaBot 384 magnetic separation stand (Promega, cat. no. V8241) 10x magnetic separation stand for 8-tube strip (10x Genomics, cat. no. 230003) DynaMag-96 side skirted magnetic separation stand (Thermo Fisher, cat. no. 12027) MagneSphere magnetic separation stand (12-hole, 1.5 mL vial, Promega, cat. no. Z5342) CoolRack XT cooling block for 384-well PCR plate (Corning, cat. no. 432055) for keeping plate cool on the deck of the Mantis liquid dispenser.

    Amplification:

    Article Title: Neoantigen vaccine generates intratumoral T cell responses in phase Ib glioblastoma trial
    Article Snippet: .. Targeted amplification of TCR transcripts was performed in a 96-well plate format using single-cell-amplified cDNA libraries (before fragmentation), either from the Smart-seq2 procedure described above or prepared using the NEBNext Single Cell/Low Input cDNA Synthesis & Amplification Module (New England BioLabs E6421L). ..

    Article Title: RNase H-dependent PCR-enabled T Cell Receptor sequencing (rhTCRseq) for Highly Specific and Efficient Targeted Sequencing of T Cell Receptor mRNA for Single-Cell and Repertoire Analysis
    Article Snippet: .. NEBNext Single Cell/Low Input cDNA Synthesis & Amplification Module (New England BioLabs, cat. no. E6421L). .. This kit contains NEBNext Cell Lysis Buffer (10×), Murine RNase Inhibitor, NEBNext Single Cell RT Primer Mix, NEBNext Single Cell RT Buffer (4×), NEBNext Template Switching Oligo, NEBNext Single Cell RT Enzyme Mix, NEBNext Single Cell cDNA PCR Master Mix (2×), and NEBNext Single Cell cDNA PCR Primer.

    Article Title: RNase H-dependent PCR-enabled T Cell Receptor sequencing (rhTCRseq) for Highly Specific and Efficient Targeted Sequencing of T Cell Receptor mRNA for Single-Cell and Repertoire Analysis
    Article Snippet: .. This protocol generates single-cell cDNA libraries in 96- or 384-well plates using reagents from the NEBNext Single Cell/Low Input cDNA Synthesis & Amplification Module (New England BioLabs E6421L). .. Steps 1A(x-xvii) are essentially the same as for Smart-seq2 , : a RT reaction with an oligo dT primer and a template-switching oligo followed by PCR amplification of full-length cDNA using a single primer.

    Article Title: Reference genome and demographic history of the most endangered marine mammal, the vaquita
    Article Snippet: .. Briefly, cDNA was reverse transcribed using the NEBNext® Single Cell/Low Input cDNA Synthesis & Amplification Module (NEB E6421S) from 238 ng total RNA. .. Amplified cDNA was cleaned with 86 μl ProNex beads.

    Article Title: Diploid chromosome-scale assembly of the Muscadinia rotundifolia genome supports chromosome fusion and disease resistance gene expansion during Vitis and Muscadinia divergence
    Article Snippet: .. First strand synthesis and cDNA amplification were accomplished using the NEBNext Single Cell/Low Input cDNA Synthesis & Amplification Module (New England, Ipswich, MA, US). .. These cDNAs were subsequently purified with ProNex magnetic beads (Promega, WI) following the instructions in the Iso-Seq Express Template Preparation for Sequel and Sequel II Systems protocol (Pacific Biosciences, Menlo Park, CA).

    Article Title: Notch ligand Dll4 impairs cell recruitment into aortic clusters and limits hematopoietic stem cells
    Article Snippet: .. Different subpopulations were directly sorted in a total volume of 10 μl of reaction buffer and processed for obtaining cDNA following manufacture’s protocol. cDNA amplification was performed by Long Distance PCR (LD-PCR) and the PCR-amplified cDNA purified by immobilization on AMPure XP beads (Agencourt AMPure XP kit). .. Samples were analyzed with Agilent High Sensitivity DNA Kit (Agilent, Cat. No. 5067-4626).

    Polymerase Chain Reaction:

    Article Title: Notch ligand Dll4 impairs cell recruitment into aortic clusters and limits hematopoietic stem cells
    Article Snippet: .. Different subpopulations were directly sorted in a total volume of 10 μl of reaction buffer and processed for obtaining cDNA following manufacture’s protocol. cDNA amplification was performed by Long Distance PCR (LD-PCR) and the PCR-amplified cDNA purified by immobilization on AMPure XP beads (Agencourt AMPure XP kit). .. Samples were analyzed with Agilent High Sensitivity DNA Kit (Agilent, Cat. No. 5067-4626).

    Purification:

    Article Title: Notch ligand Dll4 impairs cell recruitment into aortic clusters and limits hematopoietic stem cells
    Article Snippet: .. Different subpopulations were directly sorted in a total volume of 10 μl of reaction buffer and processed for obtaining cDNA following manufacture’s protocol. cDNA amplification was performed by Long Distance PCR (LD-PCR) and the PCR-amplified cDNA purified by immobilization on AMPure XP beads (Agencourt AMPure XP kit). .. Samples were analyzed with Agilent High Sensitivity DNA Kit (Agilent, Cat. No. 5067-4626).

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  • 94
    New England Biolabs input dna
    Chromatin structures at CTCF sites around the β-globin locus having deletion of GATA-1 binding motifs. ChIP was performed with antibodies specific to CTCF (A), Rad21 (B), histone H3 (C) and <t>H3K27ac</t> (D) in control cells (Con) and HS3ΔΔGA and HS2ΔGA clones. Amounts of immunoprecipitated <t>DNA</t> were determined as described in Fig 1C . The mouse Necdin gene served as an internal control. The results are the means of four independent experiments ± SEM. *P
    Input Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 114 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/input dna/product/New England Biolabs
    Average 94 stars, based on 114 article reviews
    Price from $9.99 to $1999.99
    input dna - by Bioz Stars, 2020-07
    94/100 stars
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    90
    New England Biolabs input bacmid dna
    Subcellular localization of total actin and F-actin in Sf9 cells transfected with different recombinant viruses. (A) Total actin localization. Sf9 cells were transfected with vAc54KO, vAc54:2HA, or v38KKO <t>bacmid</t> <t>DNA.</t> At 36 h p.t., the cells were fixed,
    Input Bacmid Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/input bacmid dna/product/New England Biolabs
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    input bacmid dna - by Bioz Stars, 2020-07
    90/100 stars
      Buy from Supplier

    Image Search Results


    Chromatin structures at CTCF sites around the β-globin locus having deletion of GATA-1 binding motifs. ChIP was performed with antibodies specific to CTCF (A), Rad21 (B), histone H3 (C) and H3K27ac (D) in control cells (Con) and HS3ΔΔGA and HS2ΔGA clones. Amounts of immunoprecipitated DNA were determined as described in Fig 1C . The mouse Necdin gene served as an internal control. The results are the means of four independent experiments ± SEM. *P

    Journal: bioRxiv

    Article Title: GATA-1-dependent histone H3K27ac mediates erythroid cell-specific interaction between CTCF sites

    doi: 10.1101/2020.05.14.095547

    Figure Lengend Snippet: Chromatin structures at CTCF sites around the β-globin locus having deletion of GATA-1 binding motifs. ChIP was performed with antibodies specific to CTCF (A), Rad21 (B), histone H3 (C) and H3K27ac (D) in control cells (Con) and HS3ΔΔGA and HS2ΔGA clones. Amounts of immunoprecipitated DNA were determined as described in Fig 1C . The mouse Necdin gene served as an internal control. The results are the means of four independent experiments ± SEM. *P

    Article Snippet: ChIP DNA library preparation and sequencing ChIP DNA and input DNA (10 ng for H3K27ac, 0.5 ng for CTCF and 10 ng for input) were processed using NEBNext ChIP-seq library (New England Biolabs) with manufacturer’s instructions.

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Clone Assay, Immunoprecipitation

    Chromatin interaction between CTCF sites around the β-globin locus in TSA treated MEL/ch11 cells. (A) MEL/ch11 cells were treated with 25 ng/ml of TSA for 6 h or 24 h. Histone H3 acetylated at K27 was detected by Western blotting in nuclear extract from control cells (Con) and cells treated with TSA. Histone H3 was used as an experimental control. (B) H3K27ac was determined in CTCF sites around the β-globin locus by ChIP. DNA immunoprecipitated by H3K27ac antibodies were quantitatively compared with DNA immunoprecipitated by H3, and then normalized to value in control cells. Normal IgG (IgG) was used as an experimental negative control. (C) Relative cross-linking frequencies was determined between CTCF sites around the β-globin locus in 3C assay as described in Fig 1E . Fragments containing C5 and C3 were used as anchors. Occupancies of CTCF (D) and Rad21 (E) were determined at the CTCF sites by ChIP. Results are presented as the means ± SEM of four to six independent experiments in ChIP and 3C assay. *P

    Journal: bioRxiv

    Article Title: GATA-1-dependent histone H3K27ac mediates erythroid cell-specific interaction between CTCF sites

    doi: 10.1101/2020.05.14.095547

    Figure Lengend Snippet: Chromatin interaction between CTCF sites around the β-globin locus in TSA treated MEL/ch11 cells. (A) MEL/ch11 cells were treated with 25 ng/ml of TSA for 6 h or 24 h. Histone H3 acetylated at K27 was detected by Western blotting in nuclear extract from control cells (Con) and cells treated with TSA. Histone H3 was used as an experimental control. (B) H3K27ac was determined in CTCF sites around the β-globin locus by ChIP. DNA immunoprecipitated by H3K27ac antibodies were quantitatively compared with DNA immunoprecipitated by H3, and then normalized to value in control cells. Normal IgG (IgG) was used as an experimental negative control. (C) Relative cross-linking frequencies was determined between CTCF sites around the β-globin locus in 3C assay as described in Fig 1E . Fragments containing C5 and C3 were used as anchors. Occupancies of CTCF (D) and Rad21 (E) were determined at the CTCF sites by ChIP. Results are presented as the means ± SEM of four to six independent experiments in ChIP and 3C assay. *P

    Article Snippet: ChIP DNA library preparation and sequencing ChIP DNA and input DNA (10 ng for H3K27ac, 0.5 ng for CTCF and 10 ng for input) were processed using NEBNext ChIP-seq library (New England Biolabs) with manufacturer’s instructions.

    Techniques: Western Blot, Chromatin Immunoprecipitation, Immunoprecipitation, Negative Control

    Timelines. We compared the method of Brown et al. with the results of this study obtained with Illumina MiSeq and MiniSeq and ONT MinION. We assumed that no step of the process can be initiated after 6 p.m. or before 8 a.m. The method of Brown et al. has a rapid extraction step but also a 20-h overnight enrichment step, resulting in a 50-h turnaround time. In our study, we did 27-h MiSeq runs (paired 150-bp reads), but since Mykrobe is k-mer based, a 16-h run (paired 75-bp reads) would give equivalent results; we therefore display that potential timeline here. The DNA extraction process was updated for the MiniSeq and MinION experiments, removing the ethanol precipitation step. In normal use, this would take 3 h. The 1.5-h orange rectangle on the MinION time lines includes both PCR and the 10-min sample preparation step. In this experiment, since we used spiked BCG DNA in sputum, we did not use a human depletion step, thus taking only 2 h. This image is intended to show comparable real-use time lines, and so the MiniSeq/MinION time lines are shown with 3-h extraction steps. MiniSeq enables a 16-h turnaround time by sequencing for only 7 h. R9 MinION also delivers sub-24-h results but requires one flow cell per sample. R9.4 MinION gives a 12.5-h turnaround time (6 h of sequencing with real-time [i.e., simultaneous] base calling when used on a single sample).

    Journal: Journal of Clinical Microbiology

    Article Title: Same-Day Diagnostic and Surveillance Data for Tuberculosis via Whole-Genome Sequencing of Direct Respiratory Samples

    doi: 10.1128/JCM.02483-16

    Figure Lengend Snippet: Timelines. We compared the method of Brown et al. with the results of this study obtained with Illumina MiSeq and MiniSeq and ONT MinION. We assumed that no step of the process can be initiated after 6 p.m. or before 8 a.m. The method of Brown et al. has a rapid extraction step but also a 20-h overnight enrichment step, resulting in a 50-h turnaround time. In our study, we did 27-h MiSeq runs (paired 150-bp reads), but since Mykrobe is k-mer based, a 16-h run (paired 75-bp reads) would give equivalent results; we therefore display that potential timeline here. The DNA extraction process was updated for the MiniSeq and MinION experiments, removing the ethanol precipitation step. In normal use, this would take 3 h. The 1.5-h orange rectangle on the MinION time lines includes both PCR and the 10-min sample preparation step. In this experiment, since we used spiked BCG DNA in sputum, we did not use a human depletion step, thus taking only 2 h. This image is intended to show comparable real-use time lines, and so the MiniSeq/MinION time lines are shown with 3-h extraction steps. MiniSeq enables a 16-h turnaround time by sequencing for only 7 h. R9 MinION also delivers sub-24-h results but requires one flow cell per sample. R9.4 MinION gives a 12.5-h turnaround time (6 h of sequencing with real-time [i.e., simultaneous] base calling when used on a single sample).

    Article Snippet: These samples, along with pure BCG DNA, were prepared in accordance with ONTs PCR-based protocol for low-input libraries (DP006_revB_14Aug2015), with modified primers supplied by ONT, a 20-ng DNA input into the PCR, and LongAmp Taq 2× master mix (New England BioLabs, USA).

    Techniques: DNA Extraction, Ethanol Precipitation, Polymerase Chain Reaction, Sample Prep, Sequencing, Flow Cytometry

    Attenuating nuclease activity of SpCas9. ( a ) Four PAM-interacting amino acids neighboring the nGG PAM (magenta) in the structure of SpCas9 7 . Arginines at positions 1333 and 1335 were mutated to attenuate DNA-binding affinity of SpCas9. ( b ) Activity profile of SpCas9 (blue) or SpCas9-Zif268 (red) bearing lysine or serine substitutions at positions 1333 or 1335 in the PAM interaction domain in comparison to wild-type (WT) SpCas9. Reporter assays were performed in HEK293T cells. Bar heights represent means from three independent biological replicates performed on different days. Error bars indicate standard error of the mean. ( c ) T7 Endonuclease I (T7EI) assays on PCR products spanning a genomic target site (underlined) with an NGG PAM (magenta) and neighboring Zif268 site (orange) for SpCas9 or SpCas9 mutants with or without a Zif268 fusion. For SpCas9 MT2 SpCas9 MT3 , robust nuclease activity is only observed when Zif268 is fused to the C-terminus. The gel image is representative of T7EI assays at this genomic target site, where cleaved products are noted by magenta arrowheads. ( d ) Quantification of average T7EI-based lesion rates at the PLXNB2 locus from three independent biological replicates performed on different days in HEK293T cells ( Supplementary Fig. 7 ). Error bars indicate standard error of the mean.

    Journal: Nature methods

    Article Title: DNA-binding domain fusions enhance the targeting range and precision of Cas9

    doi: 10.1038/nmeth.3624

    Figure Lengend Snippet: Attenuating nuclease activity of SpCas9. ( a ) Four PAM-interacting amino acids neighboring the nGG PAM (magenta) in the structure of SpCas9 7 . Arginines at positions 1333 and 1335 were mutated to attenuate DNA-binding affinity of SpCas9. ( b ) Activity profile of SpCas9 (blue) or SpCas9-Zif268 (red) bearing lysine or serine substitutions at positions 1333 or 1335 in the PAM interaction domain in comparison to wild-type (WT) SpCas9. Reporter assays were performed in HEK293T cells. Bar heights represent means from three independent biological replicates performed on different days. Error bars indicate standard error of the mean. ( c ) T7 Endonuclease I (T7EI) assays on PCR products spanning a genomic target site (underlined) with an NGG PAM (magenta) and neighboring Zif268 site (orange) for SpCas9 or SpCas9 mutants with or without a Zif268 fusion. For SpCas9 MT2 SpCas9 MT3 , robust nuclease activity is only observed when Zif268 is fused to the C-terminus. The gel image is representative of T7EI assays at this genomic target site, where cleaved products are noted by magenta arrowheads. ( d ) Quantification of average T7EI-based lesion rates at the PLXNB2 locus from three independent biological replicates performed on different days in HEK293T cells ( Supplementary Fig. 7 ). Error bars indicate standard error of the mean.

    Article Snippet: 50ng input DNA is PCR-amplified using “T7EI primers” that are specific for each genomic region ( ) with Phusion High Fidelity DNA Polymerase (New England Biolabs): (98°C, 15s; 67°C, 25s; 72°C, 18s) for 30 cycles.

    Techniques: Activity Assay, Binding Assay, Polymerase Chain Reaction

    Subcellular localization of total actin and F-actin in Sf9 cells transfected with different recombinant viruses. (A) Total actin localization. Sf9 cells were transfected with vAc54KO, vAc54:2HA, or v38KKO bacmid DNA. At 36 h p.t., the cells were fixed,

    Journal: Journal of Virology

    Article Title: The Autographa californica Multiple Nucleopolyhedrovirus ac54 Gene Is Crucial for Localization of the Major Capsid Protein VP39 at the Site of Nucleocapsid Assembly

    doi: 10.1128/JVI.02885-15

    Figure Lengend Snippet: Subcellular localization of total actin and F-actin in Sf9 cells transfected with different recombinant viruses. (A) Total actin localization. Sf9 cells were transfected with vAc54KO, vAc54:2HA, or v38KKO bacmid DNA. At 36 h p.t., the cells were fixed,

    Article Snippet: To eliminate input bacmid DNA, 1 μg of DNA was digested with 20 units of the DpnI restriction enzyme (NEB) overnight in a 40-μl reaction volume. qPCR was performed using Hot Start PCR master mix III (Chaoshi-Bio) and primers targeting a 100-bp region of the gp41 gene under conditions described previously ( ).

    Techniques: Transfection, Recombinant

    Subcellular localization of the minor capsid proteins PP78/83, BV/ODV-C42 (C42), and 38K, as indicated. Sf9 cells were transfected with vAc54KO, vAc54:2HA, or v38KKO bacmid DNA and fixed at 36 h p.t. (A) Transfected cells were incubated with antisera

    Journal: Journal of Virology

    Article Title: The Autographa californica Multiple Nucleopolyhedrovirus ac54 Gene Is Crucial for Localization of the Major Capsid Protein VP39 at the Site of Nucleocapsid Assembly

    doi: 10.1128/JVI.02885-15

    Figure Lengend Snippet: Subcellular localization of the minor capsid proteins PP78/83, BV/ODV-C42 (C42), and 38K, as indicated. Sf9 cells were transfected with vAc54KO, vAc54:2HA, or v38KKO bacmid DNA and fixed at 36 h p.t. (A) Transfected cells were incubated with antisera

    Article Snippet: To eliminate input bacmid DNA, 1 μg of DNA was digested with 20 units of the DpnI restriction enzyme (NEB) overnight in a 40-μl reaction volume. qPCR was performed using Hot Start PCR master mix III (Chaoshi-Bio) and primers targeting a 100-bp region of the gp41 gene under conditions described previously ( ).

    Techniques: Transfection, Incubation

    Subcellular localization of the major capsid protein VP39. Sf9 cells were transfected with vAc54KO (A), vAc54:2HA (B), or v38KKO (C) bacmid DNA. Transfected cells were fixed at 36 h p.t. and incubated with antisera against VP39 (rabbit) and IE-1 (mouse)

    Journal: Journal of Virology

    Article Title: The Autographa californica Multiple Nucleopolyhedrovirus ac54 Gene Is Crucial for Localization of the Major Capsid Protein VP39 at the Site of Nucleocapsid Assembly

    doi: 10.1128/JVI.02885-15

    Figure Lengend Snippet: Subcellular localization of the major capsid protein VP39. Sf9 cells were transfected with vAc54KO (A), vAc54:2HA (B), or v38KKO (C) bacmid DNA. Transfected cells were fixed at 36 h p.t. and incubated with antisera against VP39 (rabbit) and IE-1 (mouse)

    Article Snippet: To eliminate input bacmid DNA, 1 μg of DNA was digested with 20 units of the DpnI restriction enzyme (NEB) overnight in a 40-μl reaction volume. qPCR was performed using Hot Start PCR master mix III (Chaoshi-Bio) and primers targeting a 100-bp region of the gp41 gene under conditions described previously ( ).

    Techniques: Transfection, Incubation

    Characterization of the ac54 -knockout bacmid. (A) Strategy for the construction of the recombinant viruses vAc54KO, vAc54:2HA, and vAcWT. (B) Sf9 cells were transfected with vAc54KO, vAc54:2HA, and vAcWT bacmid DNA and observed at 72 h p.t. (C) Virus

    Journal: Journal of Virology

    Article Title: The Autographa californica Multiple Nucleopolyhedrovirus ac54 Gene Is Crucial for Localization of the Major Capsid Protein VP39 at the Site of Nucleocapsid Assembly

    doi: 10.1128/JVI.02885-15

    Figure Lengend Snippet: Characterization of the ac54 -knockout bacmid. (A) Strategy for the construction of the recombinant viruses vAc54KO, vAc54:2HA, and vAcWT. (B) Sf9 cells were transfected with vAc54KO, vAc54:2HA, and vAcWT bacmid DNA and observed at 72 h p.t. (C) Virus

    Article Snippet: To eliminate input bacmid DNA, 1 μg of DNA was digested with 20 units of the DpnI restriction enzyme (NEB) overnight in a 40-μl reaction volume. qPCR was performed using Hot Start PCR master mix III (Chaoshi-Bio) and primers targeting a 100-bp region of the gp41 gene under conditions described previously ( ).

    Techniques: Knock-Out, Recombinant, Transfection

    Immunoelectron microscopy analysis of Sf9 cells transfected with vAc54KO or vAcWT bacmid DNA at 72 h p.t. using antisera against different nucleocapsid structural proteins. For the experiments shown in panels A to C, Sf9 cells were transfected with vAc54KO

    Journal: Journal of Virology

    Article Title: The Autographa californica Multiple Nucleopolyhedrovirus ac54 Gene Is Crucial for Localization of the Major Capsid Protein VP39 at the Site of Nucleocapsid Assembly

    doi: 10.1128/JVI.02885-15

    Figure Lengend Snippet: Immunoelectron microscopy analysis of Sf9 cells transfected with vAc54KO or vAcWT bacmid DNA at 72 h p.t. using antisera against different nucleocapsid structural proteins. For the experiments shown in panels A to C, Sf9 cells were transfected with vAc54KO

    Article Snippet: To eliminate input bacmid DNA, 1 μg of DNA was digested with 20 units of the DpnI restriction enzyme (NEB) overnight in a 40-μl reaction volume. qPCR was performed using Hot Start PCR master mix III (Chaoshi-Bio) and primers targeting a 100-bp region of the gp41 gene under conditions described previously ( ).

    Techniques: Immuno-Electron Microscopy, Transfection