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
    Category:
    DNA Template Preparation for PCR
    Size:
    96 rxns
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    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 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    input dna - by Bioz Stars, 2021-06
    86/100 stars

    Images

    1) 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

    2) 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

    3) 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

    4) 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

    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 "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

    7) 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

    8) 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

    9) 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

    10) 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

    11) Product Images from "Allele-specific binding of ZFP57 in the epigenetic regulation of imprinted and non-imprinted monoallelic expression"

    Article Title: Allele-specific binding of ZFP57 in the epigenetic regulation of imprinted and non-imprinted monoallelic expression

    Journal: Genome Biology

    doi: 10.1186/s13059-015-0672-7

    Validation of strain-specific sites . a Independent ChIP-quantitative PCR validation of Zfp57 binding to non-imprinted monoallelic peaks. Overall enrichment levels are shown relative to negative control region ( REF ) and normalized to non-specific IgG pull-down. b Allele-specific analysis using SNP pyrosequencing of ChIP and 5mC-DIP enriched DNA showing Zfp57 binding is directed by both underlying DNA sequence and methylation status of the allele. Error bars represent standard deviation between three technical replicates
    Figure Legend Snippet: Validation of strain-specific sites . a Independent ChIP-quantitative PCR validation of Zfp57 binding to non-imprinted monoallelic peaks. Overall enrichment levels are shown relative to negative control region ( REF ) and normalized to non-specific IgG pull-down. b Allele-specific analysis using SNP pyrosequencing of ChIP and 5mC-DIP enriched DNA showing Zfp57 binding is directed by both underlying DNA sequence and methylation status of the allele. Error bars represent standard deviation between three technical replicates

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Negative Control, Sequencing, Methylation, Standard Deviation

    12) Product Images from "Comparison of Illumina versus Nanopore 16S rRNA Gene Sequencing of the Human Nasal Microbiota"

    Article Title: Comparison of Illumina versus Nanopore 16S rRNA Gene Sequencing of the Human Nasal Microbiota

    Journal: Genes

    doi: 10.3390/genes11091105

    Nasal microbiota profiles generated using nanopore and Illumina 16S rRNA gene sequencing. DNA was isolated from 57 nose swab samples, and 16S rRNA gene sequencing was performed using both Illumina ( a ) and nanopore ( b ) technologies. Each bar in the graph represents a nasal microbiota profile from a single individual. The dashed lines in ( b ) represent genera that, by default, were reported as unclassified at genus level in the EPI2ME report but were identified when next to reads with a top three blast hit with one genera (num_genus_taxid is 1); reads with a top three blast hit with two genera (num_genus_taxid is 2) were also included. A phylogenetic tree was generated by Pearson/UPGMA clustering of bacterial genera in microbiota profiles, as determined using Illumina sequencing. To compare between the two techniques, the sample order of the samples that were sequenced with the Oxford Nanopore platform was matched to the sample order of the samples that were sequenced with the Illumina platform, and the percentage of agreement was calculated for each nose swab sample ( c ). The horizontal black line in ( c ) indicates the mean percentage of agreement.
    Figure Legend Snippet: Nasal microbiota profiles generated using nanopore and Illumina 16S rRNA gene sequencing. DNA was isolated from 57 nose swab samples, and 16S rRNA gene sequencing was performed using both Illumina ( a ) and nanopore ( b ) technologies. Each bar in the graph represents a nasal microbiota profile from a single individual. The dashed lines in ( b ) represent genera that, by default, were reported as unclassified at genus level in the EPI2ME report but were identified when next to reads with a top three blast hit with one genera (num_genus_taxid is 1); reads with a top three blast hit with two genera (num_genus_taxid is 2) were also included. A phylogenetic tree was generated by Pearson/UPGMA clustering of bacterial genera in microbiota profiles, as determined using Illumina sequencing. To compare between the two techniques, the sample order of the samples that were sequenced with the Oxford Nanopore platform was matched to the sample order of the samples that were sequenced with the Illumina platform, and the percentage of agreement was calculated for each nose swab sample ( c ). The horizontal black line in ( c ) indicates the mean percentage of agreement.

    Techniques Used: Generated, Sequencing, Isolation

    Agarose gel with 16S rRNA gene amplicons. Total DNA was isolated from pure bacterial cultures in a similar manner as the isolation of DNA from the nasal swab samples; the DNA concentration was determined by picogreen and a PCR was performed as described for nanopore sequencing using equal amounts of template DNA, with the exception that 30 PCR cycli instead of 25 cycli were used.
    Figure Legend Snippet: Agarose gel with 16S rRNA gene amplicons. Total DNA was isolated from pure bacterial cultures in a similar manner as the isolation of DNA from the nasal swab samples; the DNA concentration was determined by picogreen and a PCR was performed as described for nanopore sequencing using equal amounts of template DNA, with the exception that 30 PCR cycli instead of 25 cycli were used.

    Techniques Used: Agarose Gel Electrophoresis, Isolation, Concentration Assay, Polymerase Chain Reaction, Nanopore Sequencing

    13) 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

    14) 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

    15) 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

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    Article Title: Transcriptomic and Physiological Analysis Reveal That α-Linolenic Acid Biosynthesis Responds to Early Chilling Tolerance in Pumpkin Rootstock Varieties
    Article Snippet: First-strand cDNA was synthesized using random hexamer primers and M-MLV reverse transcriptase (RNase H). .. Second-strand cDNA synthesis was subsequently performed using DNA polymerase I and RNase H. NEBNext Adaptors with hairpin loop structures were ligated for hybridization. .. The resulting cDNA library was sequenced on an Illumina HiSeq2500 platform (Oe-biotech, Shanghai) to obtain paired-end reads with a length of 150 bp.

    Nested PCR:

    Article Title: Massively parallel interrogation and mining of natively paired human TCRαβ repertoires
    Article Snippet: For nested PCR, the OE-RT-PCR product was first run on a 1.7% agarose gel and a band at 800–1200bp was excised and purified using NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Bethlehem, PA, USA). .. Nested PCR was performed using NEBNext amplification mix (New England Biolabs, Ipswich, MA, USA) to add adapters for Illumina sequencing or cloning into a mammalian expression construct. .. PCR products were run on a 1.2% agarose gel, and the 800–1100bp band was excised and purified using NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Bethlehem, PA, USA).

    Amplification:

    Article Title: Massively parallel interrogation and mining of natively paired human TCRαβ repertoires
    Article Snippet: For nested PCR, the OE-RT-PCR product was first run on a 1.7% agarose gel and a band at 800–1200bp was excised and purified using NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Bethlehem, PA, USA). .. Nested PCR was performed using NEBNext amplification mix (New England Biolabs, Ipswich, MA, USA) to add adapters for Illumina sequencing or cloning into a mammalian expression construct. .. PCR products were run on a 1.2% agarose gel, and the 800–1100bp band was excised and purified using NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Bethlehem, PA, USA).

    Article Title: Metatranscriptomics of N2-fixing cyanobacteria in the Amazon River plume
    Article Snippet: Low sequencing yield has previously been attributed to this kit , but multiple studies have reported high reproducibility ( ; ). .. Random primers were used with the Superscript III first-strand synthesis system (Invitrogen) to copy the amplified mRNA to complementary DNA (cDNA), followed by the NEBnext mRNA second-strand synthesis module (New England Biolabs). .. The QIAquick PCR purification kit (Qiagen) was used to purify the double-stranded cDNA, followed by ethanol precipitation.

    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: Thus, there is no need for a post-lysis purification step. .. 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: Identification of neural progenitor cells and their progeny reveals long distance migration in the developing octopus brain
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    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.

    Sequencing:

    Article Title: Massively parallel interrogation and mining of natively paired human TCRαβ repertoires
    Article Snippet: For nested PCR, the OE-RT-PCR product was first run on a 1.7% agarose gel and a band at 800–1200bp was excised and purified using NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Bethlehem, PA, USA). .. Nested PCR was performed using NEBNext amplification mix (New England Biolabs, Ipswich, MA, USA) to add adapters for Illumina sequencing or cloning into a mammalian expression construct. .. PCR products were run on a 1.2% agarose gel, and the 800–1100bp band was excised and purified using NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Bethlehem, PA, USA).

    Clone Assay:

    Article Title: Massively parallel interrogation and mining of natively paired human TCRαβ repertoires
    Article Snippet: For nested PCR, the OE-RT-PCR product was first run on a 1.7% agarose gel and a band at 800–1200bp was excised and purified using NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Bethlehem, PA, USA). .. Nested PCR was performed using NEBNext amplification mix (New England Biolabs, Ipswich, MA, USA) to add adapters for Illumina sequencing or cloning into a mammalian expression construct. .. PCR products were run on a 1.2% agarose gel, and the 800–1100bp band was excised and purified using NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Bethlehem, PA, USA).

    Expressing:

    Article Title: Massively parallel interrogation and mining of natively paired human TCRαβ repertoires
    Article Snippet: For nested PCR, the OE-RT-PCR product was first run on a 1.7% agarose gel and a band at 800–1200bp was excised and purified using NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Bethlehem, PA, USA). .. Nested PCR was performed using NEBNext amplification mix (New England Biolabs, Ipswich, MA, USA) to add adapters for Illumina sequencing or cloning into a mammalian expression construct. .. PCR products were run on a 1.2% agarose gel, and the 800–1100bp band was excised and purified using NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Bethlehem, PA, USA).

    Construct:

    Article Title: Massively parallel interrogation and mining of natively paired human TCRαβ repertoires
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    Synthesized:

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    New England Biolabs dna input
    Timelines and cost. We compare the method of Brown et al with the results of this study, using the Illumina MiSeq and MiniSeq, and the <t>ONT</t> MinION. We assume that no step of the process can be initiated after 6pm or before 8am. The method of Brown et al has a rapid extraction step, but also a 20 hour enrichment step, resulting in a 50 hour turn-around time, at an estimated cost of £203/sample. By contrast, our extraction method with MiSeq sequencing provides results at £94/sample. The <t>DNA</t> extraction process was updated for the MiniSeq and MinION experiments, removing the ethanol precipitation step. In normal use this would take 3 hours. The thin orange rectangle on the MinION timelines is the 10 minute 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 hours. This figure is intended to show comparable real-use timelines, and so the MiniSeq/MinION timelines are shown with 3 hour extraction steps. The MiniSeq enables a 16-hour turnaround time, by sequencing for only 7 hours. The R9 MinION also delivers sub-24 hour results, but requires one flow-cell per sample. The R9.4 MinION gives an 8 hour turnaround time (3 hours of sequencing with real-time (i.e. simultaneous) basecalling when used on a single sample). We do not display the 48 hours we actually took to basecall the data after the run, as this was in effect our error - we subsequently confirmed (on other samples) that real-time basecalling would have worked.
    Dna Input, 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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    New England Biolabs small rna library kit
    miRNAs regulate Cytoskeleton-Adhesion-Matrix (CAM) genes. (a) Left, Schematic of AGO2-HITS-CLIP. <t>miRNA-RNA</t> complexes were cross-linked to AGO2 (green) via UV light and unbound RNAs removed by RNase treatment. AGO2-RNA complexes were immunoprecipitated, and RNA was labelled with 32-P and isolated. Right, an Integrative Genomics Viewer display of HITS-CLIP reads showed the sequences aligned within 30 to 70 nt intervals (AGO2 peak) within the 3’UTR region of the representative genes. Both HUAEC and HUVEC share the most significant peaks (green) (b) Left, chart represents positional enrichment of AGO2 peaks within the human 3’UTR for HUAEC (red) and HUVEC (blue). Lines indicate the nt positional distribution of peak sequences within meta-gene analyzed 3’UTRs. Right chart shows difference in conservation scores across samples scoring using PhastCons (Wilcoxon Rank Sum Test). AGO2 peaks in HUAEC and HUVEC and binned human 3’UTRs were compared with binned 3’UTRs of 100 species. Conservation score is represented as a box plot with minimum, maximum, median and quartiles (n = 3 independent replica for HUVEC and HUAEC for a total of 383 for HUAEC, 749 for HUVEC and 125685 for Control individual value). (c) Left, Schematic shows cytoskeleton-adhesion-matrix (CAM) AGO2-regulome. AGO2-mRNA targets identified via AGO2-HIT-CLIP highlighted in green. Integrins, TALIN1 and BMPR1 proteins (brown) are part of CAM’s GO term but were not detected by AGO2-HITS-CLIP. Arrows point to downstream regulators of CAM proteins targeted by AGO2. CAM proteins and their regulators were identified by database searches ( Supplementary Fig. 1a , Supplementary tables 1 – 3 ) and manually curated for accuracy. Right chart shows interactome for 25 of the 73 AGO2-CAM genes in which a complementary MRE (7–8 nt) was identified using Target Scan v.7.0 prediction software and miRNAs were identified from AGO2-HITS-CLIP reads using miRbase ( Methods ). Color-coded boxes indicate the number of MREs identified in each of the selected CAM-gene 3’UTRs. Lines indicate interaction between MRE and miRNA family members with similar SEEDs. The mRNA-miRNA network shows high complexity, with numerous miRNAs binding one or more CAM-3’UTRs, while most CAM genes are targeted by more than one miRNA.
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    Timelines and cost. We compare the method of Brown et al with the results of this study, using the Illumina MiSeq and MiniSeq, and the ONT MinION. We assume that no step of the process can be initiated after 6pm or before 8am. The method of Brown et al has a rapid extraction step, but also a 20 hour enrichment step, resulting in a 50 hour turn-around time, at an estimated cost of £203/sample. By contrast, our extraction method with MiSeq sequencing provides results at £94/sample. The DNA extraction process was updated for the MiniSeq and MinION experiments, removing the ethanol precipitation step. In normal use this would take 3 hours. The thin orange rectangle on the MinION timelines is the 10 minute 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 hours. This figure is intended to show comparable real-use timelines, and so the MiniSeq/MinION timelines are shown with 3 hour extraction steps. The MiniSeq enables a 16-hour turnaround time, by sequencing for only 7 hours. The R9 MinION also delivers sub-24 hour results, but requires one flow-cell per sample. The R9.4 MinION gives an 8 hour turnaround time (3 hours of sequencing with real-time (i.e. simultaneous) basecalling when used on a single sample). We do not display the 48 hours we actually took to basecall the data after the run, as this was in effect our error - we subsequently confirmed (on other samples) that real-time basecalling would have worked.

    Journal: bioRxiv

    Article Title: Same-day diagnostic and surveillance data for tuberculosis via whole genome sequencing of direct respiratory samples

    doi: 10.1101/094789

    Figure Lengend Snippet: Timelines and cost. We compare the method of Brown et al with the results of this study, using the Illumina MiSeq and MiniSeq, and the ONT MinION. We assume that no step of the process can be initiated after 6pm or before 8am. The method of Brown et al has a rapid extraction step, but also a 20 hour enrichment step, resulting in a 50 hour turn-around time, at an estimated cost of £203/sample. By contrast, our extraction method with MiSeq sequencing provides results at £94/sample. The DNA extraction process was updated for the MiniSeq and MinION experiments, removing the ethanol precipitation step. In normal use this would take 3 hours. The thin orange rectangle on the MinION timelines is the 10 minute 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 hours. This figure is intended to show comparable real-use timelines, and so the MiniSeq/MinION timelines are shown with 3 hour extraction steps. The MiniSeq enables a 16-hour turnaround time, by sequencing for only 7 hours. The R9 MinION also delivers sub-24 hour results, but requires one flow-cell per sample. The R9.4 MinION gives an 8 hour turnaround time (3 hours of sequencing with real-time (i.e. simultaneous) basecalling when used on a single sample). We do not display the 48 hours we actually took to basecall the data after the run, as this was in effect our error - we subsequently confirmed (on other samples) that real-time basecalling would have worked.

    Article Snippet: These samples, along with pure BCG DNA, were prepared following ONTs PCR-based protocol for low-input libraries (DP006_revB_14Aug2015), using modified primers supplied by ONT, a 20 ng DNA input into the PCR reaction, and LongAmp Taq 2X Master Mix (New England Biolabs, USA).

    Techniques: Sequencing, DNA Extraction, Ethanol Precipitation, Sample Prep

    miRNAs regulate Cytoskeleton-Adhesion-Matrix (CAM) genes. (a) Left, Schematic of AGO2-HITS-CLIP. miRNA-RNA complexes were cross-linked to AGO2 (green) via UV light and unbound RNAs removed by RNase treatment. AGO2-RNA complexes were immunoprecipitated, and RNA was labelled with 32-P and isolated. Right, an Integrative Genomics Viewer display of HITS-CLIP reads showed the sequences aligned within 30 to 70 nt intervals (AGO2 peak) within the 3’UTR region of the representative genes. Both HUAEC and HUVEC share the most significant peaks (green) (b) Left, chart represents positional enrichment of AGO2 peaks within the human 3’UTR for HUAEC (red) and HUVEC (blue). Lines indicate the nt positional distribution of peak sequences within meta-gene analyzed 3’UTRs. Right chart shows difference in conservation scores across samples scoring using PhastCons (Wilcoxon Rank Sum Test). AGO2 peaks in HUAEC and HUVEC and binned human 3’UTRs were compared with binned 3’UTRs of 100 species. Conservation score is represented as a box plot with minimum, maximum, median and quartiles (n = 3 independent replica for HUVEC and HUAEC for a total of 383 for HUAEC, 749 for HUVEC and 125685 for Control individual value). (c) Left, Schematic shows cytoskeleton-adhesion-matrix (CAM) AGO2-regulome. AGO2-mRNA targets identified via AGO2-HIT-CLIP highlighted in green. Integrins, TALIN1 and BMPR1 proteins (brown) are part of CAM’s GO term but were not detected by AGO2-HITS-CLIP. Arrows point to downstream regulators of CAM proteins targeted by AGO2. CAM proteins and their regulators were identified by database searches ( Supplementary Fig. 1a , Supplementary tables 1 – 3 ) and manually curated for accuracy. Right chart shows interactome for 25 of the 73 AGO2-CAM genes in which a complementary MRE (7–8 nt) was identified using Target Scan v.7.0 prediction software and miRNAs were identified from AGO2-HITS-CLIP reads using miRbase ( Methods ). Color-coded boxes indicate the number of MREs identified in each of the selected CAM-gene 3’UTRs. Lines indicate interaction between MRE and miRNA family members with similar SEEDs. The mRNA-miRNA network shows high complexity, with numerous miRNAs binding one or more CAM-3’UTRs, while most CAM genes are targeted by more than one miRNA.

    Journal: Nature cell biology

    Article Title: microRNA-dependent regulation of biomechanical genes establishes tissue stiffness homeostasis

    doi: 10.1038/s41556-019-0272-y

    Figure Lengend Snippet: miRNAs regulate Cytoskeleton-Adhesion-Matrix (CAM) genes. (a) Left, Schematic of AGO2-HITS-CLIP. miRNA-RNA complexes were cross-linked to AGO2 (green) via UV light and unbound RNAs removed by RNase treatment. AGO2-RNA complexes were immunoprecipitated, and RNA was labelled with 32-P and isolated. Right, an Integrative Genomics Viewer display of HITS-CLIP reads showed the sequences aligned within 30 to 70 nt intervals (AGO2 peak) within the 3’UTR region of the representative genes. Both HUAEC and HUVEC share the most significant peaks (green) (b) Left, chart represents positional enrichment of AGO2 peaks within the human 3’UTR for HUAEC (red) and HUVEC (blue). Lines indicate the nt positional distribution of peak sequences within meta-gene analyzed 3’UTRs. Right chart shows difference in conservation scores across samples scoring using PhastCons (Wilcoxon Rank Sum Test). AGO2 peaks in HUAEC and HUVEC and binned human 3’UTRs were compared with binned 3’UTRs of 100 species. Conservation score is represented as a box plot with minimum, maximum, median and quartiles (n = 3 independent replica for HUVEC and HUAEC for a total of 383 for HUAEC, 749 for HUVEC and 125685 for Control individual value). (c) Left, Schematic shows cytoskeleton-adhesion-matrix (CAM) AGO2-regulome. AGO2-mRNA targets identified via AGO2-HIT-CLIP highlighted in green. Integrins, TALIN1 and BMPR1 proteins (brown) are part of CAM’s GO term but were not detected by AGO2-HITS-CLIP. Arrows point to downstream regulators of CAM proteins targeted by AGO2. CAM proteins and their regulators were identified by database searches ( Supplementary Fig. 1a , Supplementary tables 1 – 3 ) and manually curated for accuracy. Right chart shows interactome for 25 of the 73 AGO2-CAM genes in which a complementary MRE (7–8 nt) was identified using Target Scan v.7.0 prediction software and miRNAs were identified from AGO2-HITS-CLIP reads using miRbase ( Methods ). Color-coded boxes indicate the number of MREs identified in each of the selected CAM-gene 3’UTRs. Lines indicate interaction between MRE and miRNA family members with similar SEEDs. The mRNA-miRNA network shows high complexity, with numerous miRNAs binding one or more CAM-3’UTRs, while most CAM genes are targeted by more than one miRNA.

    Article Snippet: Libraries were amplified with 12 PCR cycles. miRNA libraries were prepared from 1μg of total RNA using the NEBNext® Small RNA Library Kit (NEB) following the gel size selection method in manufacturer’s protocol and submitted for Illumina sequencing.

    Techniques: Chick Chorioallantoic Membrane Assay, Cross-linking Immunoprecipitation, Immunoprecipitation, Isolation, Software, Binding Assay

    Illustration of the three scRNA-seq protocols applied in this study. A) NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina (NEB®) followed by NEBNext® uracil excision based (UEB) Final Library Preparation KIT. B) SMART-seq® High-Throughput (HT) kit (Takara Bio Inc.) followed by final library preparation using Nextera XT Library preparation kit (Illumina, USA). C) Genome transcriptome sequencing (G T-seq) followed by final library preparation using Nextera XT Library preparation kit (Illumina, USA).

    Journal: bioRxiv

    Article Title: Benchmarking full-length transcript single cell mRNA sequencing protocols

    doi: 10.1101/2020.07.29.225201

    Figure Lengend Snippet: Illustration of the three scRNA-seq protocols applied in this study. A) NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina (NEB®) followed by NEBNext® uracil excision based (UEB) Final Library Preparation KIT. B) SMART-seq® High-Throughput (HT) kit (Takara Bio Inc.) followed by final library preparation using Nextera XT Library preparation kit (Illumina, USA). C) Genome transcriptome sequencing (G T-seq) followed by final library preparation using Nextera XT Library preparation kit (Illumina, USA).

    Article Snippet: NEBNext® Single Cell/ Low Input RNA Library Prep Kit 14 single cells were processed using NEBNext® Single Cell/ Low Input RNA Library Prep Kit (cat nr.

    Techniques: High Throughput Screening Assay, Sequencing