ultra dna library prep kit  (New England Biolabs)


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    NEBNext Ultra DNA Library Prep Kit for Illumina
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    NEBNext Ultra DNA Library Prep Kit for Illumina 96 rxns
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    e7370l
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    96 rxns
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    DNA Template Preparation for PCR
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    New England Biolabs ultra dna library prep kit
    NEBNext Ultra DNA Library Prep Kit for Illumina
    NEBNext Ultra DNA Library Prep Kit for Illumina 96 rxns
    https://www.bioz.com/result/ultra dna library prep kit/product/New England Biolabs
    Average 95 stars, based on 201 article reviews
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    ultra dna library prep kit - by Bioz Stars, 2020-02
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    1) Product Images from "A Method for Selectively Enriching Microbial DNA from Contaminating Vertebrate Host DNA"

    Article Title: A Method for Selectively Enriching Microbial DNA from Contaminating Vertebrate Host DNA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0076096

    Analysis of Illumina sequence reads of a black molly ( Poecilia cf. sphenops ) whole fish DNA library before and after enrichment with MBD-Fc protein A paramagnetic beads. Shown is a concordance plot comparing relative abundances of microbial genera between the enriched and unenriched samples.
    Figure Legend Snippet: Analysis of Illumina sequence reads of a black molly ( Poecilia cf. sphenops ) whole fish DNA library before and after enrichment with MBD-Fc protein A paramagnetic beads. Shown is a concordance plot comparing relative abundances of microbial genera between the enriched and unenriched samples.

    Techniques Used: Sequencing, Fluorescence In Situ Hybridization

    MBD-Fc separates human DNA from human-malarial DNA mixtures. (A) Graph of the percentage of 75 bp Illumina reads mapping to the Plasmodium falciparum reference sequence in the mixture before enrichment (Unenriched), after enrichment (Enriched) and the bound fraction following wash and elution as described above (Bound). (B) Evenness of coverage analysis metrics show enriched malaria reads (Enriched Plasmodium ) in line with pure malaria DNA reads ( Plasmodium Only). Unlike the unenriched input sample (Human + Plasmodium ) showing 60% of the Plasmodium genome uncovered (zero depth), enriched sample (Enriched Plasmodium ) showed even coverage of the genome with no regions lacking coverage. The amount of Plasmodium DNA retained in the pellet (Bound Human) following wash and elution was insignificant. (C) GC-content and bias analysis. No base bias was detected in the enriched sample. Average GC content of the enriched sample (Enriched Plasmodium ) matches the pure malaria sample ( Plasmodium Only) and is very close to the theoretical GC coverage (Theoretical).
    Figure Legend Snippet: MBD-Fc separates human DNA from human-malarial DNA mixtures. (A) Graph of the percentage of 75 bp Illumina reads mapping to the Plasmodium falciparum reference sequence in the mixture before enrichment (Unenriched), after enrichment (Enriched) and the bound fraction following wash and elution as described above (Bound). (B) Evenness of coverage analysis metrics show enriched malaria reads (Enriched Plasmodium ) in line with pure malaria DNA reads ( Plasmodium Only). Unlike the unenriched input sample (Human + Plasmodium ) showing 60% of the Plasmodium genome uncovered (zero depth), enriched sample (Enriched Plasmodium ) showed even coverage of the genome with no regions lacking coverage. The amount of Plasmodium DNA retained in the pellet (Bound Human) following wash and elution was insignificant. (C) GC-content and bias analysis. No base bias was detected in the enriched sample. Average GC content of the enriched sample (Enriched Plasmodium ) matches the pure malaria sample ( Plasmodium Only) and is very close to the theoretical GC coverage (Theoretical).

    Techniques Used: Sequencing

    2) Product Images from "A Comprehensive Analysis of Replicating Merkel Cell Polyomavirus Genomes Delineates the Viral Transcription Program and Suggests a Role for mcv-miR-M1 in Episomal Persistence"

    Article Title: A Comprehensive Analysis of Replicating Merkel Cell Polyomavirus Genomes Delineates the Viral Transcription Program and Suggests a Role for mcv-miR-M1 in Episomal Persistence

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004974

    RCA analysis suggests episomal persistence of MCVSyn genomes. Inverted image of a ethidium bromide-stained agarose gel with input material (lanes 1,3,5,7 and 9) or RCA products (lanes 2,4,6,8, 10 and 11) from mock-transfected PFSK-1 cells (lanes 1 and 2), MCVSyn-transfected PFSK-1 cells at 4 days (lanes 3 and 4) or 136 days (lanes 9 and 10) post transfection, the MCC-derived cell lines WaGa or MKL-1 (lanes 5 and 6 or 7 and 8, respectively) or a water control (lane 11). DNA was subjected to restriction enzyme digestion to produce linear, unit-length viral genomes from concatameric RCA products. For size comparison, lane 12 shows unit length MCVSyn genomes excised from plasmid pMK-MCVSyn. The asterisk marks the position of the bacterial pMK plasmid backbone.
    Figure Legend Snippet: RCA analysis suggests episomal persistence of MCVSyn genomes. Inverted image of a ethidium bromide-stained agarose gel with input material (lanes 1,3,5,7 and 9) or RCA products (lanes 2,4,6,8, 10 and 11) from mock-transfected PFSK-1 cells (lanes 1 and 2), MCVSyn-transfected PFSK-1 cells at 4 days (lanes 3 and 4) or 136 days (lanes 9 and 10) post transfection, the MCC-derived cell lines WaGa or MKL-1 (lanes 5 and 6 or 7 and 8, respectively) or a water control (lane 11). DNA was subjected to restriction enzyme digestion to produce linear, unit-length viral genomes from concatameric RCA products. For size comparison, lane 12 shows unit length MCVSyn genomes excised from plasmid pMK-MCVSyn. The asterisk marks the position of the bacterial pMK plasmid backbone.

    Techniques Used: Staining, Agarose Gel Electrophoresis, Transfection, Derivative Assay, Plasmid Preparation

    3) Product Images from "From the Cover: Germline-restricted chromosome (GRC) is widespread among songbirds"

    Article Title: From the Cover: Germline-restricted chromosome (GRC) is widespread among songbirds

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1817373116

    Discovery of GRCs in bird species. ( A ) SC spreads of four oscine species immunolabeled with antibodies against SYCP3, the main protein of the lateral element of SC (red), centromere proteins (blue) and MLH1, mismatch repair protein marking recombination sites (green). Arrowheads point to the largest chromosomes ordered according to their size ranks, ZZ (identified by its size and arm ratio), ZW (identified by heteromorphic SC and misaligned centromeres), and GRCs. Arrows in the Insets point to MLH1 foci in GRCs. Micro-GRC bivalents in female barn swallow and European pied flycatcher are indistinguishable from the microchromosomes of the somatic chromosome set. ( B ) DAPI-stained bone marrow cells. ( C ) Reverse and cross-species FISH of GRC DNA probes (green) derived from Bengalese finch (LST), zebra finch (TGU), Eurasian siskin (SSP), and pale martin (RDI) with SC spreads, immunolabeled with antibodies against SYCP3 (red). Centromeres are labeled with antibodies against centromere proteins (blue). Arrowheads point to GRCs and regions on the somatic chromosome set intensely painted with GRC probes in cross-species FISH. Insets show GRCs. The Bengalese finch GRC-specific DNA probe gives a strong signal on the Bengalese finch GRC and slightly paints some regions of the somatic chromosome set. The zebra finch GRC probe paints the distal area of the Bengalese finch GRC and a region of the short arm of SC3. The Eurasian siskin GRC probe paints a micro-GRC of European goldfinch, a region on the long arm of SC3 and some pericentromeric regions. The pale martin GRC probe gives a dispersed signal on the great tit GRC, the ZW bivalent and on SC5. Scale bar: 5 µm.
    Figure Legend Snippet: Discovery of GRCs in bird species. ( A ) SC spreads of four oscine species immunolabeled with antibodies against SYCP3, the main protein of the lateral element of SC (red), centromere proteins (blue) and MLH1, mismatch repair protein marking recombination sites (green). Arrowheads point to the largest chromosomes ordered according to their size ranks, ZZ (identified by its size and arm ratio), ZW (identified by heteromorphic SC and misaligned centromeres), and GRCs. Arrows in the Insets point to MLH1 foci in GRCs. Micro-GRC bivalents in female barn swallow and European pied flycatcher are indistinguishable from the microchromosomes of the somatic chromosome set. ( B ) DAPI-stained bone marrow cells. ( C ) Reverse and cross-species FISH of GRC DNA probes (green) derived from Bengalese finch (LST), zebra finch (TGU), Eurasian siskin (SSP), and pale martin (RDI) with SC spreads, immunolabeled with antibodies against SYCP3 (red). Centromeres are labeled with antibodies against centromere proteins (blue). Arrowheads point to GRCs and regions on the somatic chromosome set intensely painted with GRC probes in cross-species FISH. Insets show GRCs. The Bengalese finch GRC-specific DNA probe gives a strong signal on the Bengalese finch GRC and slightly paints some regions of the somatic chromosome set. The zebra finch GRC probe paints the distal area of the Bengalese finch GRC and a region of the short arm of SC3. The Eurasian siskin GRC probe paints a micro-GRC of European goldfinch, a region on the long arm of SC3 and some pericentromeric regions. The pale martin GRC probe gives a dispersed signal on the great tit GRC, the ZW bivalent and on SC5. Scale bar: 5 µm.

    Techniques Used: Immunolabeling, Staining, Fluorescence In Situ Hybridization, Derivative Assay, Labeling

    4) Product Images from "Histone H3.3 sub-variant H3mm7 is required for normal skeletal muscle regeneration"

    Article Title: Histone H3.3 sub-variant H3mm7 is required for normal skeletal muscle regeneration

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03845-1

    Ala57 allows the mobility of the H3mm7 nucleosome. a T32I and S57A are only the differences between H3.3 and H3mm7. N-terminals of the amino acid sequence of H3.3 and H3mm7 are shown in the illustration of the secondary structure. b High-resolution crystal structure of an H3mm7-containing nucleosome shows the loss of interaction between H3.3S57 and H4R40 in the H3mm7 nucleosome. An overview of the crystal structure of the H3mm7-containing nucleosome (left) and the comparative view around S57 in H3.3 and A57 in H3mm7 (right). c H3mm7 nucleosomes were dissociated at a concentration of 600 mM NaCl. The salt-titration assay of H3mm7, H3.3, and the mutant (H3.3S57A and T32I) containing nucleosomes is shown. The positions of the bands that correspond with the intact nucleosome core particle and naked DNA are indicated on the right. d H3.3S57A and H3mm7 dissociated from the H2A:H2B dimer faster than the other H3 variant-containing nucleosomes. Thermal stability assay of H3mm7, H3.3, and the point mutants H3.3T32I and H3.3S57A. The lines show the fluorescent intensity that indicates nucleosome dissociation at each temperature. The error bars are ± 1 SD ( n = 4). e H3mm7 and H3.3S57A showed a fast histone turnover. FRAP analysis of nucleosomes containing GFP-fused H3mm7, H3.3, and the point mutants. Representative confocal images of FRAP analysis in each H3 variant nucleosome is shown on the left. Plot of average GFP fluorescent recovery rate at each time point after photobleaching is shown on the right. The error bars are ± 1 SD. The number of replicates is indicated in parentheses
    Figure Legend Snippet: Ala57 allows the mobility of the H3mm7 nucleosome. a T32I and S57A are only the differences between H3.3 and H3mm7. N-terminals of the amino acid sequence of H3.3 and H3mm7 are shown in the illustration of the secondary structure. b High-resolution crystal structure of an H3mm7-containing nucleosome shows the loss of interaction between H3.3S57 and H4R40 in the H3mm7 nucleosome. An overview of the crystal structure of the H3mm7-containing nucleosome (left) and the comparative view around S57 in H3.3 and A57 in H3mm7 (right). c H3mm7 nucleosomes were dissociated at a concentration of 600 mM NaCl. The salt-titration assay of H3mm7, H3.3, and the mutant (H3.3S57A and T32I) containing nucleosomes is shown. The positions of the bands that correspond with the intact nucleosome core particle and naked DNA are indicated on the right. d H3.3S57A and H3mm7 dissociated from the H2A:H2B dimer faster than the other H3 variant-containing nucleosomes. Thermal stability assay of H3mm7, H3.3, and the point mutants H3.3T32I and H3.3S57A. The lines show the fluorescent intensity that indicates nucleosome dissociation at each temperature. The error bars are ± 1 SD ( n = 4). e H3mm7 and H3.3S57A showed a fast histone turnover. FRAP analysis of nucleosomes containing GFP-fused H3mm7, H3.3, and the point mutants. Representative confocal images of FRAP analysis in each H3 variant nucleosome is shown on the left. Plot of average GFP fluorescent recovery rate at each time point after photobleaching is shown on the right. The error bars are ± 1 SD. The number of replicates is indicated in parentheses

    Techniques Used: Sequencing, Concentration Assay, Titration, Mutagenesis, Variant Assay, Stability Assay

    5) Product Images from "Evidence for L1-associated DNA rearrangements and negligible L1 retrotransposition in glioblastoma multiforme"

    Article Title: Evidence for L1-associated DNA rearrangements and negligible L1 retrotransposition in glioblastoma multiforme

    Journal: Mobile DNA

    doi: 10.1186/s13100-016-0076-6

    Characterisation of a somatic L1 mutation within EGFR. a Patient #8 EGFR mutant allele: a 0.5 kb L1-Ta sequence antisense to EGFR. Direction of transcription is indicated with blue arrow . b L1 mutation magnified view: RC-seq reads detected at the L1 3' terminus ( black/red bars ). The L1 mutation comprised a truncated fragment of L1 ORF2 ( white box ) and the 3′UTR without a poly-A tail ( red box ). A 550 nucleotide deletion at the integration site was also identified (triangle). Primers used for PCR validation are indicated as pink and purple arrows. c Mutation site PCR validation: Region comprising the EGFR-L1 5' junction was detected in patient #8 tumour sample. No amplification was detected when water (NTC) or genomic DNA from blood were used as template. d qRT-PCR measurement of EGFR transcription at its 5'UTR and exon 11-to-12 junction (E 11–12): The relative levels of RNA from both regions were significantly increased in tumour ( blue ) versus adjacent brain ( green ) samples. Data for each group were normalised to adjacent brain values, pooled and presented as mean +/− SEM (* p
    Figure Legend Snippet: Characterisation of a somatic L1 mutation within EGFR. a Patient #8 EGFR mutant allele: a 0.5 kb L1-Ta sequence antisense to EGFR. Direction of transcription is indicated with blue arrow . b L1 mutation magnified view: RC-seq reads detected at the L1 3' terminus ( black/red bars ). The L1 mutation comprised a truncated fragment of L1 ORF2 ( white box ) and the 3′UTR without a poly-A tail ( red box ). A 550 nucleotide deletion at the integration site was also identified (triangle). Primers used for PCR validation are indicated as pink and purple arrows. c Mutation site PCR validation: Region comprising the EGFR-L1 5' junction was detected in patient #8 tumour sample. No amplification was detected when water (NTC) or genomic DNA from blood were used as template. d qRT-PCR measurement of EGFR transcription at its 5'UTR and exon 11-to-12 junction (E 11–12): The relative levels of RNA from both regions were significantly increased in tumour ( blue ) versus adjacent brain ( green ) samples. Data for each group were normalised to adjacent brain values, pooled and presented as mean +/− SEM (* p

    Techniques Used: Mutagenesis, Sequencing, Polymerase Chain Reaction, Amplification, Quantitative RT-PCR

    Characterisation of a somatic L1-associated DNA rearrangement within MeCP2. a Patient #2 MeCP2 mutant allele: a 0.9 kb L1PA2 sequence antisense to MeCP2. Direction of transcription ( blue arrows ), transcript isoforms ( purple/pink lines ) and qRT-PCR primers for MeCP2 expression assays ( arrowheads ) are indicated. b L1 mutation magnified view: RC-seq reads detected at the L1 5′ terminus ( black/white bars ). The L1 sequence comprises a truncated fragment of L1 ORF2 ( white box ), the 3′UTR without a poly-A tail ( red box ) and 37 nt from an Alu ( black box ). A 58 nucleotide deletion was also identified (triangle). Primers used for PCR validation are indicated as grey arrows. c Mutation site PCR validation: the mutant MeCP2 allele carrying L1 (filled) was only detected in patient #2 tumour whilst the empty site was found in both tumour and adjacent brain samples. No amplification was detected when water was used as template (NTC). d qRT-PCR measurement of MeCP2 transcript isoforms: The relative levels of RNA from both isoforms were significantly reduced in tumour ( blue ) versus adjacent brain ( green ) samples. Data for each group were normalised to non-tumour values, pooled and presented as mean +/− SEM (* p
    Figure Legend Snippet: Characterisation of a somatic L1-associated DNA rearrangement within MeCP2. a Patient #2 MeCP2 mutant allele: a 0.9 kb L1PA2 sequence antisense to MeCP2. Direction of transcription ( blue arrows ), transcript isoforms ( purple/pink lines ) and qRT-PCR primers for MeCP2 expression assays ( arrowheads ) are indicated. b L1 mutation magnified view: RC-seq reads detected at the L1 5′ terminus ( black/white bars ). The L1 sequence comprises a truncated fragment of L1 ORF2 ( white box ), the 3′UTR without a poly-A tail ( red box ) and 37 nt from an Alu ( black box ). A 58 nucleotide deletion was also identified (triangle). Primers used for PCR validation are indicated as grey arrows. c Mutation site PCR validation: the mutant MeCP2 allele carrying L1 (filled) was only detected in patient #2 tumour whilst the empty site was found in both tumour and adjacent brain samples. No amplification was detected when water was used as template (NTC). d qRT-PCR measurement of MeCP2 transcript isoforms: The relative levels of RNA from both isoforms were significantly reduced in tumour ( blue ) versus adjacent brain ( green ) samples. Data for each group were normalised to non-tumour values, pooled and presented as mean +/− SEM (* p

    Techniques Used: Mutagenesis, Sequencing, Quantitative RT-PCR, Expressing, Polymerase Chain Reaction, Amplification

    6) Product Images from "A high throughput screen for active human transposable elements"

    Article Title: A high throughput screen for active human transposable elements

    Journal: BMC Genomics

    doi: 10.1186/s12864-018-4485-4

    TE-NGS sequencing workflow. Enrichment for genomic fragments spanning active TEs and their unique flanking sequence is achieved by several enzymatic steps as described in the main text. First, genomic DNA is sheared, and adapters for sequencing are ligated to the genomic fragments following standard library preparation protocols. Next, a small aliquot (10 ng) of library is used as template for targeted amplification with primers complementary to TE subfamily-specific sequences and to the Illumina Universal PCR (P5) primer. Remaining genomic background fragments and inverted TEs in head-to-head orientation are removed by ssDNA exonuclease digestion after linear PCR amplification with TE-target primers or Illumina Universal primer, respectively. Last, amplification with nested primers targeting TE diagnostic bases, and containing Illumina i7 index and P7 primer sequences generates full double-stranded dual-adapter libraries containing unique indices for each sample and each TE subfamily, allowing for downstream pooling and multiplexing of many samples simultaneously. High throughput sequencing followed by alignment to the reference genome demarcates the TE insertion site by its 3′ end (read 2) and unique flanking sequence (read 1). TE insertions present in the reference genome can be identified by clustering of read pairs, whereas read 2 generated from polymorphic or novel TE insertions absent from the reference will map with lower quality and/or not at all; these TE can be identified by clusters of read 1 alone (see Methods; Supplemental Material for detailed procedures)
    Figure Legend Snippet: TE-NGS sequencing workflow. Enrichment for genomic fragments spanning active TEs and their unique flanking sequence is achieved by several enzymatic steps as described in the main text. First, genomic DNA is sheared, and adapters for sequencing are ligated to the genomic fragments following standard library preparation protocols. Next, a small aliquot (10 ng) of library is used as template for targeted amplification with primers complementary to TE subfamily-specific sequences and to the Illumina Universal PCR (P5) primer. Remaining genomic background fragments and inverted TEs in head-to-head orientation are removed by ssDNA exonuclease digestion after linear PCR amplification with TE-target primers or Illumina Universal primer, respectively. Last, amplification with nested primers targeting TE diagnostic bases, and containing Illumina i7 index and P7 primer sequences generates full double-stranded dual-adapter libraries containing unique indices for each sample and each TE subfamily, allowing for downstream pooling and multiplexing of many samples simultaneously. High throughput sequencing followed by alignment to the reference genome demarcates the TE insertion site by its 3′ end (read 2) and unique flanking sequence (read 1). TE insertions present in the reference genome can be identified by clustering of read pairs, whereas read 2 generated from polymorphic or novel TE insertions absent from the reference will map with lower quality and/or not at all; these TE can be identified by clusters of read 1 alone (see Methods; Supplemental Material for detailed procedures)

    Techniques Used: Next-Generation Sequencing, Sequencing, Amplification, Polymerase Chain Reaction, Diagnostic Assay, Multiplexing, Generated

    7) Product Images from "A Comprehensive Analysis of Replicating Merkel Cell Polyomavirus Genomes Delineates the Viral Transcription Program and Suggests a Role for mcv-miR-M1 in Episomal Persistence"

    Article Title: A Comprehensive Analysis of Replicating Merkel Cell Polyomavirus Genomes Delineates the Viral Transcription Program and Suggests a Role for mcv-miR-M1 in Episomal Persistence

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004974

    ChIP-seq analysis and miRNA expression in wt and mutated MCVSyn genomes. ( A ) Schematic depiction of the MCPyV genome with polyadenylation and transcriptional initiation sites as identified by RACE analyses. The position of the viral core origin of replication (ori) is marked by a grey box. ( B, E ) ChIP-seq profiles of LT-Ag (top panel, blue), H3K4me3 (center panel, green) or the negative IgG control (bottom panel, grey) along the MCPyV genome in PFSK-1 cells after 4 days of transfection with MCVSyn ( B ) or MCVSyn-pmt ( E ). Positions of early and late transcriptional initiation sites are marked by bent arrows pointing right and left, respectively. The putative promoter region mutated in MCVSyn-pmt is symbolized by a vertically hatched box in E. Graphs depict raw ChIP-seq read coverage; note that absolute read numbers depend on the efficiency of individual immunoprecipitations and thus only the relative coverage distribution along the viral genome is meaningful. ( C ) Mutations in the putative promoter region upstream of the mcv-miR-M1 locus. Substituted nucleotides are shown in red. The positions of the transcriptional initiation site TI-L2 is indicated by an arrow. The first four nucleotides of the mcv-miR-M1 locus are boxed in gray. (D) Quantitative stem-loop RT-PCR evaluation of mcv-miR-M1-5p expression in PFSK-1 cells after 4 days of transfection with MCVSyn (left), MCVSyn-pmt (center), or MCVSyn-hpko (negative control, right). Expression levels were normalized to the number of MCVSyn genomes per cell as determined by qPCR from genomic DNA. Mean values and standard deviations were calculated from three independent experiments. mcv-miR-M1 expression in MCVSyn-pmt is significantly decreased in comparison to MCVSyn (unpaired t-test).
    Figure Legend Snippet: ChIP-seq analysis and miRNA expression in wt and mutated MCVSyn genomes. ( A ) Schematic depiction of the MCPyV genome with polyadenylation and transcriptional initiation sites as identified by RACE analyses. The position of the viral core origin of replication (ori) is marked by a grey box. ( B, E ) ChIP-seq profiles of LT-Ag (top panel, blue), H3K4me3 (center panel, green) or the negative IgG control (bottom panel, grey) along the MCPyV genome in PFSK-1 cells after 4 days of transfection with MCVSyn ( B ) or MCVSyn-pmt ( E ). Positions of early and late transcriptional initiation sites are marked by bent arrows pointing right and left, respectively. The putative promoter region mutated in MCVSyn-pmt is symbolized by a vertically hatched box in E. Graphs depict raw ChIP-seq read coverage; note that absolute read numbers depend on the efficiency of individual immunoprecipitations and thus only the relative coverage distribution along the viral genome is meaningful. ( C ) Mutations in the putative promoter region upstream of the mcv-miR-M1 locus. Substituted nucleotides are shown in red. The positions of the transcriptional initiation site TI-L2 is indicated by an arrow. The first four nucleotides of the mcv-miR-M1 locus are boxed in gray. (D) Quantitative stem-loop RT-PCR evaluation of mcv-miR-M1-5p expression in PFSK-1 cells after 4 days of transfection with MCVSyn (left), MCVSyn-pmt (center), or MCVSyn-hpko (negative control, right). Expression levels were normalized to the number of MCVSyn genomes per cell as determined by qPCR from genomic DNA. Mean values and standard deviations were calculated from three independent experiments. mcv-miR-M1 expression in MCVSyn-pmt is significantly decreased in comparison to MCVSyn (unpaired t-test).

    Techniques Used: Chromatin Immunoprecipitation, Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Negative Control, Real-time Polymerase Chain Reaction

    8) Product Images from "Structural conservation in a membrane-enveloped filamentous virus infecting a hyperthermophilic acidophile"

    Article Title: Structural conservation in a membrane-enveloped filamentous virus infecting a hyperthermophilic acidophile

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05684-6

    Structural conservation and divergence among capsid proteins. Heterodimers ( a , b , c , e ) and homodimers ( d ) form the capsids in SFV1 ( a , b , c ), SIRV2 ( d ) and AFV1 ( e ). a , b VP5 (red) and VP4 (yellow) from SFV1 have been aligned to each other. The long α-helix that wraps around the DNA is structurally quite conserved between the two. The main differences between the two coat proteins is that VP5 has an insert (from ∼141 to 169) that contains the two β-strands seen in ( b ), and VP5 has a C-terminal extension that contains two short helices. This C-terminal extension crosses the helical groove and makes a large contact with a subunit in the next turn. The SFV1 heterodimer ( c ) is more similar in how it wraps DNA to the SIRV2 homodimer ( d ) than it is to the AFV1 heterodimer ( e )
    Figure Legend Snippet: Structural conservation and divergence among capsid proteins. Heterodimers ( a , b , c , e ) and homodimers ( d ) form the capsids in SFV1 ( a , b , c ), SIRV2 ( d ) and AFV1 ( e ). a , b VP5 (red) and VP4 (yellow) from SFV1 have been aligned to each other. The long α-helix that wraps around the DNA is structurally quite conserved between the two. The main differences between the two coat proteins is that VP5 has an insert (from ∼141 to 169) that contains the two β-strands seen in ( b ), and VP5 has a C-terminal extension that contains two short helices. This C-terminal extension crosses the helical groove and makes a large contact with a subunit in the next turn. The SFV1 heterodimer ( c ) is more similar in how it wraps DNA to the SIRV2 homodimer ( d ) than it is to the AFV1 heterodimer ( e )

    Techniques Used:

    Three-dimensional reconstruction of SFV1. A top view ( a ) shows the membrane enveloping the nucleoprotein core. The membrane has been filtered to 7 Å resolution and cylindrically averaged. The nucleoprotein core has been filtered to 3.7 Å resolution. In the side view ( b ) the membrane has been removed. c A view from within the lumen of the virion shows the atomic model built into the density. The asymmetric unit contains VP5 (red), VP4 (yellow) and 12 basepairs of DNA. One strand of DNA is shown in magenta, the other in cyan. VP5 contains a small C-terminal domain that makes an extensive contact with a subunit in the helical turn above. d The radial density profile of the virion is shown, after cylindrically averaging the structure. Two slightly different estimates of the solvent density come from the inside and the outside of the structure. The higher density on the outside could arise from bound ions or glycosylation, particularly since it has been determined that the membrane protein VP3 is glycosylated (Supplementary Fig. 2 )
    Figure Legend Snippet: Three-dimensional reconstruction of SFV1. A top view ( a ) shows the membrane enveloping the nucleoprotein core. The membrane has been filtered to 7 Å resolution and cylindrically averaged. The nucleoprotein core has been filtered to 3.7 Å resolution. In the side view ( b ) the membrane has been removed. c A view from within the lumen of the virion shows the atomic model built into the density. The asymmetric unit contains VP5 (red), VP4 (yellow) and 12 basepairs of DNA. One strand of DNA is shown in magenta, the other in cyan. VP5 contains a small C-terminal domain that makes an extensive contact with a subunit in the helical turn above. d The radial density profile of the virion is shown, after cylindrically averaging the structure. Two slightly different estimates of the solvent density come from the inside and the outside of the structure. The higher density on the outside could arise from bound ions or glycosylation, particularly since it has been determined that the membrane protein VP3 is glycosylated (Supplementary Fig. 2 )

    Techniques Used:

    9) Product Images from "Silencio/CG9754 connects the Piwi–piRNA complex to the cellular heterochromatin machinery"

    Article Title: Silencio/CG9754 connects the Piwi–piRNA complex to the cellular heterochromatin machinery

    Journal: Genes & Development

    doi: 10.1101/gad.271908.115

    CG9754 elicits transcriptional silencing upon recruitment to nascent RNA or DNA. ( A ) The cartoon depicts the GFP-5xboxB reporter used for RNA tethering. It harbors the ubiquitous α-tubulin promoter, the GFP-coding sequence, and a 3′ UTR harboring five boxB sites. The confocal image below depicts GFP fluorescence signal in an egg chamber expressing the GFP-boxB reporter. Throughout this study, we converted GFP signal to monochrome images with an inverted black/white color scheme for displaying optimal contrast. Soma and germline tissue is indicated. Images at the right depict the expression of λN-HA fusion proteins under control of the germline-specific MTD-Gal4 driver (λN-GW182: cytoplasmic; λN-Piwi: nuclear-enriched). ( B ) GFP fluorescence in egg chambers expressing the ubiquitous GFP-boxB reporter and the indicated λN fusions in the germline. ( C ) Simplified cartoon of the linear piRNA pathway feeding into Piwi, also indicating key players involved at the indicated steps. (TGS) Transcriptional gene silencing. ( D ) GFP fluorescence in egg chambers expressing the ubiquitous GFP-boxB reporter and the indicated λN fusions in the germline. Expression of LacI-CG9754 served as a negative control. ( E ) Shown are changes in GFP-boxB steady-state RNA levels in ovaries expressing the indicated λN fusions (normalized to rp49 ). n = 3. Error bars indicate SD. ( F ) Shown is RNA Pol II occupancy at the indicated loci determined by chromatin immunoprecipitation (ChIP) followed by quantitative PCR using ovaries expressing the indicated λN fusions. n = 3. Error bars indicate SD. Signals were normalized to those at the TSS of the act 5C gene. arrestin is not expressed in ovaries. ( G ) The cartoon depicts the 8xlacO-GFP reporter used for DNA tethering. This reporter is expressed in germline and somatic cells of the ovary ( nanos promoter coupled to intronic piwi ). The confocal image below depicts GFP fluorescence signal of the reporter. The middle image depicts the expression of LacI-CG9754 under control of the germline-specific MTD-Gal4 driver. Images at the right show GFP fluorescence signal of the reporter in egg chambers expressing the indicated LacI fusions in the germline. Note that LacI-Piwi accumulates mostly in the cytoplasm (potentially due to the reported oligomerization of LacI) (Supplemental Fig. S1E) and thus cannot be interpreted unambiguously. ( H ) Shown are GFP fluorescence signals in egg chambers expressing the GFP-boxB reporter, λN-CG9754, and the indicated shRNA constructs in the germline.
    Figure Legend Snippet: CG9754 elicits transcriptional silencing upon recruitment to nascent RNA or DNA. ( A ) The cartoon depicts the GFP-5xboxB reporter used for RNA tethering. It harbors the ubiquitous α-tubulin promoter, the GFP-coding sequence, and a 3′ UTR harboring five boxB sites. The confocal image below depicts GFP fluorescence signal in an egg chamber expressing the GFP-boxB reporter. Throughout this study, we converted GFP signal to monochrome images with an inverted black/white color scheme for displaying optimal contrast. Soma and germline tissue is indicated. Images at the right depict the expression of λN-HA fusion proteins under control of the germline-specific MTD-Gal4 driver (λN-GW182: cytoplasmic; λN-Piwi: nuclear-enriched). ( B ) GFP fluorescence in egg chambers expressing the ubiquitous GFP-boxB reporter and the indicated λN fusions in the germline. ( C ) Simplified cartoon of the linear piRNA pathway feeding into Piwi, also indicating key players involved at the indicated steps. (TGS) Transcriptional gene silencing. ( D ) GFP fluorescence in egg chambers expressing the ubiquitous GFP-boxB reporter and the indicated λN fusions in the germline. Expression of LacI-CG9754 served as a negative control. ( E ) Shown are changes in GFP-boxB steady-state RNA levels in ovaries expressing the indicated λN fusions (normalized to rp49 ). n = 3. Error bars indicate SD. ( F ) Shown is RNA Pol II occupancy at the indicated loci determined by chromatin immunoprecipitation (ChIP) followed by quantitative PCR using ovaries expressing the indicated λN fusions. n = 3. Error bars indicate SD. Signals were normalized to those at the TSS of the act 5C gene. arrestin is not expressed in ovaries. ( G ) The cartoon depicts the 8xlacO-GFP reporter used for DNA tethering. This reporter is expressed in germline and somatic cells of the ovary ( nanos promoter coupled to intronic piwi ). The confocal image below depicts GFP fluorescence signal of the reporter. The middle image depicts the expression of LacI-CG9754 under control of the germline-specific MTD-Gal4 driver. Images at the right show GFP fluorescence signal of the reporter in egg chambers expressing the indicated LacI fusions in the germline. Note that LacI-Piwi accumulates mostly in the cytoplasm (potentially due to the reported oligomerization of LacI) (Supplemental Fig. S1E) and thus cannot be interpreted unambiguously. ( H ) Shown are GFP fluorescence signals in egg chambers expressing the GFP-boxB reporter, λN-CG9754, and the indicated shRNA constructs in the germline.

    Techniques Used: Sequencing, Fluorescence, Expressing, Negative Control, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Activated Clotting Time Assay, shRNA, Construct

    10) Product Images from "An NGS-based approach for the identification of sex-specific markers in snakehead (Channa argus)"

    Article Title: An NGS-based approach for the identification of sex-specific markers in snakehead (Channa argus)

    Journal: Oncotarget

    doi: 10.18632/oncotarget.21924

    NGS data of snakeheads and analysis data during screening the sex-specific molecular markers DNAs of M1, F1, F’-Mix were used for DNA sequencing. Libraries were separately run in three lane of an Illumina HiSeq 2000, using 150 base paired-end reads. The raw reads were filtered, and obtained clean reads. 196 candidate Y chromosome-specific fragments were screened from the NGS data through bioinformatics analysis, such as genome assembly and alignments.
    Figure Legend Snippet: NGS data of snakeheads and analysis data during screening the sex-specific molecular markers DNAs of M1, F1, F’-Mix were used for DNA sequencing. Libraries were separately run in three lane of an Illumina HiSeq 2000, using 150 base paired-end reads. The raw reads were filtered, and obtained clean reads. 196 candidate Y chromosome-specific fragments were screened from the NGS data through bioinformatics analysis, such as genome assembly and alignments.

    Techniques Used: Next-Generation Sequencing, DNA Sequencing

    11) Product Images from "Unliganded Progesterone Receptor Governs Estrogen Receptor Gene Expression by Regulating DNA Methylation in Breast Cancer Cells"

    Article Title: Unliganded Progesterone Receptor Governs Estrogen Receptor Gene Expression by Regulating DNA Methylation in Breast Cancer Cells

    Journal: Cancers

    doi: 10.3390/cancers10100371

    DNA methylation affects PR binding to the ESR1 promoter. ( a ) The high-methylated ESR1 promoter, in contrast to the low-methylated intronic sequence, was only partially bound by PR in PR-rescued cells (T47D-Y + PR). ChIP assays were performed with a specific antibody against PR. Specific binding was assessed by quantitative PCR amplification of the ESR1 gene promoter and an enhancer-like intronic sequence in T47D control cells, PR-deficient cells (T47D-Y) and PR-rescued cells (T47D-Y + PR). Error bars represent the SD of three independent experiments. * p less than or equal to 0.05, ** p less than or equal to 0.01, *** p less than or equal to 0.005, unpaired two-tailed Student’s t -test. ( b ) The 5-azacytidine (5-azaC) demethylated ESR1 promoter. DNA methylation analysis was performed by the MeDIP-qPCR assay using T47D-Y + PR cells treated with the demethylating agent 5-azaC (5 µM) or vehicle (control). The results are represented as fold change relative to the control. Error bars represent the SD of three independent experiments. ** p less than or equal to 0.01. ( c ) The demethylating agent 5-azaC increases PR binding at the ESR1 promoter in PR-rescued cells (T47D-Y + PR). ChIP was performed as in (b) using T47D-Y + PR cells treated for 112 h with the demethylating agent 5-azaC (5 µM) or vehicle (control). Error bars represent the SD of three independent experiments. p = 0.07, unpaired two-tailed Student’s t -test.
    Figure Legend Snippet: DNA methylation affects PR binding to the ESR1 promoter. ( a ) The high-methylated ESR1 promoter, in contrast to the low-methylated intronic sequence, was only partially bound by PR in PR-rescued cells (T47D-Y + PR). ChIP assays were performed with a specific antibody against PR. Specific binding was assessed by quantitative PCR amplification of the ESR1 gene promoter and an enhancer-like intronic sequence in T47D control cells, PR-deficient cells (T47D-Y) and PR-rescued cells (T47D-Y + PR). Error bars represent the SD of three independent experiments. * p less than or equal to 0.05, ** p less than or equal to 0.01, *** p less than or equal to 0.005, unpaired two-tailed Student’s t -test. ( b ) The 5-azacytidine (5-azaC) demethylated ESR1 promoter. DNA methylation analysis was performed by the MeDIP-qPCR assay using T47D-Y + PR cells treated with the demethylating agent 5-azaC (5 µM) or vehicle (control). The results are represented as fold change relative to the control. Error bars represent the SD of three independent experiments. ** p less than or equal to 0.01. ( c ) The demethylating agent 5-azaC increases PR binding at the ESR1 promoter in PR-rescued cells (T47D-Y + PR). ChIP was performed as in (b) using T47D-Y + PR cells treated for 112 h with the demethylating agent 5-azaC (5 µM) or vehicle (control). Error bars represent the SD of three independent experiments. p = 0.07, unpaired two-tailed Student’s t -test.

    Techniques Used: DNA Methylation Assay, Binding Assay, Methylation, Sequencing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Amplification, Two Tailed Test, Methylated DNA Immunoprecipitation

    The loss of PR increases the DNA methylation level at the ESR1 promoter. DNA methylation of the ESR1 promoter and the enhancer-like intron was assessed by methylated DNA immunoprecipitation (MeDIP)-qPCR in T47D control cells, T47D-Y cells and T47D-Y cells with stable PR transfection (T47D-Y + PR) ( a ), or in T47D cells transduced with shRNA against PR (shPR; clone trcn0000010776) or scrambled shRNA (shC) ( b ). The results are represented as values relative to the control (T47D or shC). IgG, negative control for immunoprecipitation. Error bars represent the SD of three independent experiments. ** p less than or equal to 0.01, unpaired two-tailed Student’s t -test.
    Figure Legend Snippet: The loss of PR increases the DNA methylation level at the ESR1 promoter. DNA methylation of the ESR1 promoter and the enhancer-like intron was assessed by methylated DNA immunoprecipitation (MeDIP)-qPCR in T47D control cells, T47D-Y cells and T47D-Y cells with stable PR transfection (T47D-Y + PR) ( a ), or in T47D cells transduced with shRNA against PR (shPR; clone trcn0000010776) or scrambled shRNA (shC) ( b ). The results are represented as values relative to the control (T47D or shC). IgG, negative control for immunoprecipitation. Error bars represent the SD of three independent experiments. ** p less than or equal to 0.01, unpaired two-tailed Student’s t -test.

    Techniques Used: DNA Methylation Assay, Methylation, Immunoprecipitation, Methylated DNA Immunoprecipitation, Real-time Polymerase Chain Reaction, Transfection, Transduction, shRNA, Negative Control, Two Tailed Test

    12) Product Images from "Histone H3.3 sub-variant H3mm7 is required for normal skeletal muscle regeneration"

    Article Title: Histone H3.3 sub-variant H3mm7 is required for normal skeletal muscle regeneration

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03845-1

    Ala57 allows the mobility of the H3mm7 nucleosome. a T32I and S57A are only the differences between H3.3 and H3mm7. N-terminals of the amino acid sequence of H3.3 and H3mm7 are shown in the illustration of the secondary structure. b High-resolution crystal structure of an H3mm7-containing nucleosome shows the loss of interaction between H3.3S57 and H4R40 in the H3mm7 nucleosome. An overview of the crystal structure of the H3mm7-containing nucleosome (left) and the comparative view around S57 in H3.3 and A57 in H3mm7 (right). c H3mm7 nucleosomes were dissociated at a concentration of 600 mM NaCl. The salt-titration assay of H3mm7, H3.3, and the mutant (H3.3S57A and T32I) containing nucleosomes is shown. The positions of the bands that correspond with the intact nucleosome core particle and naked DNA are indicated on the right. d H3.3S57A and H3mm7 dissociated from the H2A:H2B dimer faster than the other H3 variant-containing nucleosomes. Thermal stability assay of H3mm7, H3.3, and the point mutants H3.3T32I and H3.3S57A. The lines show the fluorescent intensity that indicates nucleosome dissociation at each temperature. The error bars are ± 1 SD ( n = 4). e H3mm7 and H3.3S57A showed a fast histone turnover. FRAP analysis of nucleosomes containing GFP-fused H3mm7, H3.3, and the point mutants. Representative confocal images of FRAP analysis in each H3 variant nucleosome is shown on the left. Plot of average GFP fluorescent recovery rate at each time point after photobleaching is shown on the right. The error bars are ± 1 SD. The number of replicates is indicated in parentheses
    Figure Legend Snippet: Ala57 allows the mobility of the H3mm7 nucleosome. a T32I and S57A are only the differences between H3.3 and H3mm7. N-terminals of the amino acid sequence of H3.3 and H3mm7 are shown in the illustration of the secondary structure. b High-resolution crystal structure of an H3mm7-containing nucleosome shows the loss of interaction between H3.3S57 and H4R40 in the H3mm7 nucleosome. An overview of the crystal structure of the H3mm7-containing nucleosome (left) and the comparative view around S57 in H3.3 and A57 in H3mm7 (right). c H3mm7 nucleosomes were dissociated at a concentration of 600 mM NaCl. The salt-titration assay of H3mm7, H3.3, and the mutant (H3.3S57A and T32I) containing nucleosomes is shown. The positions of the bands that correspond with the intact nucleosome core particle and naked DNA are indicated on the right. d H3.3S57A and H3mm7 dissociated from the H2A:H2B dimer faster than the other H3 variant-containing nucleosomes. Thermal stability assay of H3mm7, H3.3, and the point mutants H3.3T32I and H3.3S57A. The lines show the fluorescent intensity that indicates nucleosome dissociation at each temperature. The error bars are ± 1 SD ( n = 4). e H3mm7 and H3.3S57A showed a fast histone turnover. FRAP analysis of nucleosomes containing GFP-fused H3mm7, H3.3, and the point mutants. Representative confocal images of FRAP analysis in each H3 variant nucleosome is shown on the left. Plot of average GFP fluorescent recovery rate at each time point after photobleaching is shown on the right. The error bars are ± 1 SD. The number of replicates is indicated in parentheses

    Techniques Used: Sequencing, Concentration Assay, Titration, Mutagenesis, Variant Assay, Stability Assay

    13) Product Images from "Reference Materials for Calibration of Analytical Biases in Quantification of DNA Methylation"

    Article Title: Reference Materials for Calibration of Analytical Biases in Quantification of DNA Methylation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0137006

    Comparative validation of methylation controls from commercial sources with the synthetic reference materials. Methylation levels of commercial DNA methylation controls and synthetic reference materials were measured by melting- ( A ) and NGS-based analyses ( B ).
    Figure Legend Snippet: Comparative validation of methylation controls from commercial sources with the synthetic reference materials. Methylation levels of commercial DNA methylation controls and synthetic reference materials were measured by melting- ( A ) and NGS-based analyses ( B ).

    Techniques Used: Methylation, DNA Methylation Assay, Next-Generation Sequencing

    Gene-dependent PCR biases in DNA methylation analyses. Normalized melting profiles (left), standard curves from melting analyses (center) and standard curves from NGS analyses (right) from analysis of working standards of P14 ( A ), P16 ( B ) and MLH1 ( C ) genes. Standard curves for NGS analyses were plotted based on the read counts for unmethylated and methylated DNA from amplicon sequencing as provided in the S5 Table . No PCR bias for P14 , preferential amplification of unmethylated templates for P16 and preferential amplification of methylated templates for MLH1 were consistently observed from both melting- and NGS-based analyses. Bias values (b) were calculated based on a previously described equation [ 19 ].
    Figure Legend Snippet: Gene-dependent PCR biases in DNA methylation analyses. Normalized melting profiles (left), standard curves from melting analyses (center) and standard curves from NGS analyses (right) from analysis of working standards of P14 ( A ), P16 ( B ) and MLH1 ( C ) genes. Standard curves for NGS analyses were plotted based on the read counts for unmethylated and methylated DNA from amplicon sequencing as provided in the S5 Table . No PCR bias for P14 , preferential amplification of unmethylated templates for P16 and preferential amplification of methylated templates for MLH1 were consistently observed from both melting- and NGS-based analyses. Bias values (b) were calculated based on a previously described equation [ 19 ].

    Techniques Used: Polymerase Chain Reaction, DNA Methylation Assay, Next-Generation Sequencing, Methylation, Amplification, Sequencing

    14) Product Images from "Bead-linked transposomes enable a normalization-free workflow for NGS library preparation"

    Article Title: Bead-linked transposomes enable a normalization-free workflow for NGS library preparation

    Journal: BMC Genomics

    doi: 10.1186/s12864-018-5096-9

    Improved coverage of human and bacterial genomes with Nextera DNA Flex. a Coverage across important regions of the human genome by three library preparation kits: Nextera DNA Flex, TruSeq Nano, and TruSeq PCR-free. b Coverage across extreme regions of the human genome by three library preparation kits: Nextera DNA Flex, TruSeq Nano, and TruSeq PCR-free. c Libraries generated from the small genomes of bacteria with low ( B. cereus ), medium ( E. coli ), and high ( R. sphaeroides ) GC content at 1 ng inputs. Less coverage variation was observed with libraries prepared by Nextera DNA Flex compared with libraries prepared by other commercially available library preparation kits (NEB, NEBNext Ultra DNA Library Prep Kit; Kapa A, Kapa HyperPlus Kits; Kapa B, Kapa HyperPrep Kits), particularly for the low and high GC content genomes of B. cereus and R. sphaeroides. The method of DNA fragmentation and whether PCR amplification was used during library preparation is indicated
    Figure Legend Snippet: Improved coverage of human and bacterial genomes with Nextera DNA Flex. a Coverage across important regions of the human genome by three library preparation kits: Nextera DNA Flex, TruSeq Nano, and TruSeq PCR-free. b Coverage across extreme regions of the human genome by three library preparation kits: Nextera DNA Flex, TruSeq Nano, and TruSeq PCR-free. c Libraries generated from the small genomes of bacteria with low ( B. cereus ), medium ( E. coli ), and high ( R. sphaeroides ) GC content at 1 ng inputs. Less coverage variation was observed with libraries prepared by Nextera DNA Flex compared with libraries prepared by other commercially available library preparation kits (NEB, NEBNext Ultra DNA Library Prep Kit; Kapa A, Kapa HyperPlus Kits; Kapa B, Kapa HyperPrep Kits), particularly for the low and high GC content genomes of B. cereus and R. sphaeroides. The method of DNA fragmentation and whether PCR amplification was used during library preparation is indicated

    Techniques Used: Polymerase Chain Reaction, Generated, Amplification

    15) Product Images from "Ultrasensitive and high-efficiency screen of de novo low-frequency mutations by o2n-seq"

    Article Title: Ultrasensitive and high-efficiency screen of de novo low-frequency mutations by o2n-seq

    Journal: Nature Communications

    doi: 10.1038/ncomms15335

    Overview of o2n-seq. ( a ) O2n-seq workflow. gDNA is sheared into fragments shorter than half the length of the sequencing read (for example, if we sequence reads with PE 125, the length of fragments should shorter than 125 bp) and ligated with Y-shaped adaptors (blue and orange lines) with candidate nicking sites (such as dUTP), then denatured into single-stranded DNA molecules that are circularized using single-stranded DNA ligase. After eliminating linear DNA using DNA exonucleases, the circularized single-stranded DNA is employed for second-strand synthesis. Next, the double-stranded circular DNA is nicked with the USER enzyme and subjected to strand displacement amplification. The purified, strand displacement-amplified DNA can be used to prepare a standard NGS library. The arrows represent the direction of DNA (5′→3′). ( b ) Remove errors. If a variant (red star) is support by only one DNA copy, an error must have occurred at the site, and this type of site is discarded in the following data analysis procedure. However, if a variant is supported by both DNA copies, it is treated as a true variation. ( c ) Using a PE sequencing strategy to sequence o2n-seq reads. Read 1 and Read 2 are sequencing reads of one PE read.
    Figure Legend Snippet: Overview of o2n-seq. ( a ) O2n-seq workflow. gDNA is sheared into fragments shorter than half the length of the sequencing read (for example, if we sequence reads with PE 125, the length of fragments should shorter than 125 bp) and ligated with Y-shaped adaptors (blue and orange lines) with candidate nicking sites (such as dUTP), then denatured into single-stranded DNA molecules that are circularized using single-stranded DNA ligase. After eliminating linear DNA using DNA exonucleases, the circularized single-stranded DNA is employed for second-strand synthesis. Next, the double-stranded circular DNA is nicked with the USER enzyme and subjected to strand displacement amplification. The purified, strand displacement-amplified DNA can be used to prepare a standard NGS library. The arrows represent the direction of DNA (5′→3′). ( b ) Remove errors. If a variant (red star) is support by only one DNA copy, an error must have occurred at the site, and this type of site is discarded in the following data analysis procedure. However, if a variant is supported by both DNA copies, it is treated as a true variation. ( c ) Using a PE sequencing strategy to sequence o2n-seq reads. Read 1 and Read 2 are sequencing reads of one PE read.

    Techniques Used: Sequencing, Amplification, Purification, Next-Generation Sequencing, Variant Assay

    16) Product Images from "Bacterial artificial chromosomes establish replication timing and sub-nuclear compartment de novo as extra-chromosomal vectors"

    Article Title: Bacterial artificial chromosomes establish replication timing and sub-nuclear compartment de novo as extra-chromosomal vectors

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx1265

    E-BAC replication timing. ( A ) Experimental flowchart for Repli-seq. Exponentially growing cells were labeled with BrdU for 2 h, sorted for DNA content into early versus late S phase. DNA was isolated, sheared and the nascent DNA synthesized either early or late during S phase was immunoprecipitated with an anti-BrdU antibody. BrdU-substituted DNA was sequenced and the log 2 ratio of sequences in early versus late S phase fractions was calculated in 6 kb non-overlapping windows. To determine copy number, the reads from early and late fractions were summed to acquire whole genome sequencing information and the total number of reads per 6kb window was determined (see Figure 3B and Materials and Methods). ( B ) E-BAC repli-seq profiles. The Y-axis for each data point is the log 2 ratio for reads within a 6 kb window. HeLa-EBNA1 cells without E-BAC transfection were used as control (gray data points). Vertical pink dashed lines indicate the human chromosomal map positions of the segments contained in each BAC. RT of the 21 kb bacterial sequences of each E-BAC (pBACe3.6 E-BAC backbone) was plotted separately and indicated as horizontal lines (average of all 6kb windows) next to the chromosomal regions. (C, D) Replication timing of the EBV genome in Raji cells ( C ) and the EBV BAC in HeLa-EBNA1/EBV ( D ) (red data points). The datapoints for HeLa-EBNA1 EBV BAC are more dispersed because there are fewer total reads due to the substantially lower copy number of the EBV sequences in this cell line ( Supplementary Figure S9 ). Data from 15 Mb of chr8 is plotted for comparison (grey data points).
    Figure Legend Snippet: E-BAC replication timing. ( A ) Experimental flowchart for Repli-seq. Exponentially growing cells were labeled with BrdU for 2 h, sorted for DNA content into early versus late S phase. DNA was isolated, sheared and the nascent DNA synthesized either early or late during S phase was immunoprecipitated with an anti-BrdU antibody. BrdU-substituted DNA was sequenced and the log 2 ratio of sequences in early versus late S phase fractions was calculated in 6 kb non-overlapping windows. To determine copy number, the reads from early and late fractions were summed to acquire whole genome sequencing information and the total number of reads per 6kb window was determined (see Figure 3B and Materials and Methods). ( B ) E-BAC repli-seq profiles. The Y-axis for each data point is the log 2 ratio for reads within a 6 kb window. HeLa-EBNA1 cells without E-BAC transfection were used as control (gray data points). Vertical pink dashed lines indicate the human chromosomal map positions of the segments contained in each BAC. RT of the 21 kb bacterial sequences of each E-BAC (pBACe3.6 E-BAC backbone) was plotted separately and indicated as horizontal lines (average of all 6kb windows) next to the chromosomal regions. (C, D) Replication timing of the EBV genome in Raji cells ( C ) and the EBV BAC in HeLa-EBNA1/EBV ( D ) (red data points). The datapoints for HeLa-EBNA1 EBV BAC are more dispersed because there are fewer total reads due to the substantially lower copy number of the EBV sequences in this cell line ( Supplementary Figure S9 ). Data from 15 Mb of chr8 is plotted for comparison (grey data points).

    Techniques Used: BAC Assay, Labeling, Isolation, Synthesized, Immunoprecipitation, Sequencing, Transfection

    17) Product Images from "Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing"

    Article Title: Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing

    Journal: Nature biotechnology

    doi: 10.1038/nbt.3073

    MAB-seq strategy and quantitative mapping of active DNA demethylation ( a ) Schematic diagram of MAB-seq. DNMT methylates unmodified C to generate 5mC, which can be successively oxidized by TET to generate 5hmC/5fC/5caC. Highly oxidized cytosine derivatives, 5fC and 5caC, are repaired by TDG/BER to regenerate unmodified C. ( b ) M.SssI exhibits robust methylase activity towards unmodified cytosines within CpGs, but has significantly lower activity for cytosines in non-CpG contexts (CHG or CHH, H=A, T, C). M.SssI methylase activity was measured by MAB-seq analysis (Illumina deep sequencing) of unmethylated lambda DNA. Standard bisulfite sequencing (BS-seq) confirmed nearly complete conversion of unmethylated C to T at CpG and non-CpG sites. ( c ) Locus-specific MAB-seq analysis of 5fC/5caC at Tbx5 by Illumina sequencing in control ( shCtrl ) and Tdg knockdown ( shTdg ) mouse ESCs. For comparison, also shown are 5fC/5caC antibody DNA immunoprecipitation (DIP) based maps of 5fC and 5caC in control and Tdg -depleted mouse ESCs. DIP-seq tracks are represented in normalized read density (reads per 10 million reads, rp10m) and the vertical axis range of all DIP-seq tracks is from 1 to 25. Black horizontal bars denote 5fC/5caC-enriched regions identified by 5fC/5caC DIP-seq. The level of 5fC/5caC (only Watson strand shown) is displayed as the percentage of total C modified as 5fC/5caC, and background signals detected in Tet1/2 −/− mouse ESCs was subtracted. ( d ) Statistical calling of 5fC/5caC-modified CpGs in locus-specific MAB-seq analysis. Shown are 5fC/5caC levels (background corrected using raw MAB-seq signals in Tet1/2 −/− ) of 11 CpG sites from the Tbx5 locus in shCtrl, shCtrl +VC, shTdg and shTdg +VC mouse ESCs. shTdg was compared with shCtrl while shTdg +VC was compared with shCtrl +VC. An asterisk indicates that a CpG is statistically enriched for 5fC/5caC (multiple comparison corrected P
    Figure Legend Snippet: MAB-seq strategy and quantitative mapping of active DNA demethylation ( a ) Schematic diagram of MAB-seq. DNMT methylates unmodified C to generate 5mC, which can be successively oxidized by TET to generate 5hmC/5fC/5caC. Highly oxidized cytosine derivatives, 5fC and 5caC, are repaired by TDG/BER to regenerate unmodified C. ( b ) M.SssI exhibits robust methylase activity towards unmodified cytosines within CpGs, but has significantly lower activity for cytosines in non-CpG contexts (CHG or CHH, H=A, T, C). M.SssI methylase activity was measured by MAB-seq analysis (Illumina deep sequencing) of unmethylated lambda DNA. Standard bisulfite sequencing (BS-seq) confirmed nearly complete conversion of unmethylated C to T at CpG and non-CpG sites. ( c ) Locus-specific MAB-seq analysis of 5fC/5caC at Tbx5 by Illumina sequencing in control ( shCtrl ) and Tdg knockdown ( shTdg ) mouse ESCs. For comparison, also shown are 5fC/5caC antibody DNA immunoprecipitation (DIP) based maps of 5fC and 5caC in control and Tdg -depleted mouse ESCs. DIP-seq tracks are represented in normalized read density (reads per 10 million reads, rp10m) and the vertical axis range of all DIP-seq tracks is from 1 to 25. Black horizontal bars denote 5fC/5caC-enriched regions identified by 5fC/5caC DIP-seq. The level of 5fC/5caC (only Watson strand shown) is displayed as the percentage of total C modified as 5fC/5caC, and background signals detected in Tet1/2 −/− mouse ESCs was subtracted. ( d ) Statistical calling of 5fC/5caC-modified CpGs in locus-specific MAB-seq analysis. Shown are 5fC/5caC levels (background corrected using raw MAB-seq signals in Tet1/2 −/− ) of 11 CpG sites from the Tbx5 locus in shCtrl, shCtrl +VC, shTdg and shTdg +VC mouse ESCs. shTdg was compared with shCtrl while shTdg +VC was compared with shCtrl +VC. An asterisk indicates that a CpG is statistically enriched for 5fC/5caC (multiple comparison corrected P

    Techniques Used: Activity Assay, Sequencing, Lambda DNA Preparation, Methylation Sequencing, Immunoprecipitation, DNA Immunoprecipitation Sequencing, Modification

    18) Product Images from "Stability of patient-specific features of altered DNA replication timing in xenografts of primary human acute lymphoblastic leukemia"

    Article Title: Stability of patient-specific features of altered DNA replication timing in xenografts of primary human acute lymphoblastic leukemia

    Journal: Experimental hematology

    doi: 10.1016/j.exphem.2017.04.004

    RT profiles from ALL PDXs A. Outline of a genome-wide RT assay. BrdU labeling of nascent DNA (E/L) is preferred whenever cells are actively proliferating. However, RT profiles can also be obtained on patient samples that have lost metabolic activity but retain their S-phase DNA content based on an analysis of their DNA copy number differences (S/G1). B. Comparison of log 2 ratios for both methods applied to the same spleen cells derived from a B-ALL PDX. Although the S/G1 method gives a dynamic range of
    Figure Legend Snippet: RT profiles from ALL PDXs A. Outline of a genome-wide RT assay. BrdU labeling of nascent DNA (E/L) is preferred whenever cells are actively proliferating. However, RT profiles can also be obtained on patient samples that have lost metabolic activity but retain their S-phase DNA content based on an analysis of their DNA copy number differences (S/G1). B. Comparison of log 2 ratios for both methods applied to the same spleen cells derived from a B-ALL PDX. Although the S/G1 method gives a dynamic range of

    Techniques Used: Genome Wide, Labeling, Activity Assay, Derivative Assay

    19) Product Images from "Structural conservation in a membrane-enveloped filamentous virus infecting a hyperthermophilic acidophile"

    Article Title: Structural conservation in a membrane-enveloped filamentous virus infecting a hyperthermophilic acidophile

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05684-6

    Structural conservation and divergence among capsid proteins. Heterodimers ( a , b , c , e ) and homodimers ( d ) form the capsids in SFV1 ( a , b , c ), SIRV2 ( d ) and AFV1 ( e ). a , b VP5 (red) and VP4 (yellow) from SFV1 have been aligned to each other. The long α-helix that wraps around the DNA is structurally quite conserved between the two. The main differences between the two coat proteins is that VP5 has an insert (from ∼141 to 169) that contains the two β-strands seen in ( b ), and VP5 has a C-terminal extension that contains two short helices. This C-terminal extension crosses the helical groove and makes a large contact with a subunit in the next turn. The SFV1 heterodimer ( c ) is more similar in how it wraps DNA to the SIRV2 homodimer ( d ) than it is to the AFV1 heterodimer ( e )
    Figure Legend Snippet: Structural conservation and divergence among capsid proteins. Heterodimers ( a , b , c , e ) and homodimers ( d ) form the capsids in SFV1 ( a , b , c ), SIRV2 ( d ) and AFV1 ( e ). a , b VP5 (red) and VP4 (yellow) from SFV1 have been aligned to each other. The long α-helix that wraps around the DNA is structurally quite conserved between the two. The main differences between the two coat proteins is that VP5 has an insert (from ∼141 to 169) that contains the two β-strands seen in ( b ), and VP5 has a C-terminal extension that contains two short helices. This C-terminal extension crosses the helical groove and makes a large contact with a subunit in the next turn. The SFV1 heterodimer ( c ) is more similar in how it wraps DNA to the SIRV2 homodimer ( d ) than it is to the AFV1 heterodimer ( e )

    Techniques Used:

    Three-dimensional reconstruction of SFV1. A top view ( a ) shows the membrane enveloping the nucleoprotein core. The membrane has been filtered to 7 Å resolution and cylindrically averaged. The nucleoprotein core has been filtered to 3.7 Å resolution. In the side view ( b ) the membrane has been removed. c A view from within the lumen of the virion shows the atomic model built into the density. The asymmetric unit contains VP5 (red), VP4 (yellow) and 12 basepairs of DNA. One strand of DNA is shown in magenta, the other in cyan. VP5 contains a small C-terminal domain that makes an extensive contact with a subunit in the helical turn above. d )
    Figure Legend Snippet: Three-dimensional reconstruction of SFV1. A top view ( a ) shows the membrane enveloping the nucleoprotein core. The membrane has been filtered to 7 Å resolution and cylindrically averaged. The nucleoprotein core has been filtered to 3.7 Å resolution. In the side view ( b ) the membrane has been removed. c A view from within the lumen of the virion shows the atomic model built into the density. The asymmetric unit contains VP5 (red), VP4 (yellow) and 12 basepairs of DNA. One strand of DNA is shown in magenta, the other in cyan. VP5 contains a small C-terminal domain that makes an extensive contact with a subunit in the helical turn above. d )

    Techniques Used:

    20) Product Images from "The minimal amount of starting DNA for Agilent’s hybrid capture-based targeted massively parallel sequencing"

    Article Title: The minimal amount of starting DNA for Agilent’s hybrid capture-based targeted massively parallel sequencing

    Journal: Scientific Reports

    doi: 10.1038/srep26732

    Comparison of kits using HapMap cell line DNA ( a–c ) and FFPE samples ( d,e ). ( a ) Stacked bar plot showing subgroups of filtered reads and reads left after filtering (i.e., on-target) for five commercial kits using 200 ng, 50 ng, and 10 ng of input DNA. The NEBNext Ultra kit failed to construct an acceptable library using 200 ng of input DNA (indicated by an asterisk). ( b ) Coverage efficiency comparison using 50 ng of input DNA (n = 2). Coverage efficiency is visualized as the percent of the total targeted bases covered at particular depths. Data in ( a , b ) were averaged from two sets of libraries from two HapMap pools (10 cell lines each). ( c ) Base substitution detection sensitivity of libraries using 50 ng of input DNA was plotted against expected allele frequencies of base substitution alterations (x-axis). ( d ) Coverage efficiency comparison using an FFPE sample (FFPE1 in e ). ( e ) Comparison of base substitution concordance among libraries from the same sample. For each of four samples, three libraries were generated using 300 ng, 100 ng, or 50 ng of input DNA. Depending on the number of libraries detecting a variant, variants were classified into three groups; private to a library, shared by two libraries, or found in three libraries.
    Figure Legend Snippet: Comparison of kits using HapMap cell line DNA ( a–c ) and FFPE samples ( d,e ). ( a ) Stacked bar plot showing subgroups of filtered reads and reads left after filtering (i.e., on-target) for five commercial kits using 200 ng, 50 ng, and 10 ng of input DNA. The NEBNext Ultra kit failed to construct an acceptable library using 200 ng of input DNA (indicated by an asterisk). ( b ) Coverage efficiency comparison using 50 ng of input DNA (n = 2). Coverage efficiency is visualized as the percent of the total targeted bases covered at particular depths. Data in ( a , b ) were averaged from two sets of libraries from two HapMap pools (10 cell lines each). ( c ) Base substitution detection sensitivity of libraries using 50 ng of input DNA was plotted against expected allele frequencies of base substitution alterations (x-axis). ( d ) Coverage efficiency comparison using an FFPE sample (FFPE1 in e ). ( e ) Comparison of base substitution concordance among libraries from the same sample. For each of four samples, three libraries were generated using 300 ng, 100 ng, or 50 ng of input DNA. Depending on the number of libraries detecting a variant, variants were classified into three groups; private to a library, shared by two libraries, or found in three libraries.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Construct, Generated, Variant Assay

    21) Product Images from "Chromosomal Instability Estimation Based on Next Generation Sequencing and Single Cell Genome Wide Copy Number Variation Analysis"

    Article Title: Chromosomal Instability Estimation Based on Next Generation Sequencing and Single Cell Genome Wide Copy Number Variation Analysis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0165089

    Whole Genome Amplification, library preparation quality control, read quality, output, and alignment quality. (A) DNA concentrations and total yield for each single cell WGA as measured by UV/Vis. Libraries were constructed from independent replicates of single cells: 8 from LNCaP, 8 from PC3, 5 from VCaP, and 4 from WBCs. Overall, we achieved an average 100% success rate during the single cell whole genome amplification procedure: 8/8 single cells (100%) successfully amplified for PC3 and LNCaP cancer cell lines, while 5/5 (100%) single VCaP cells and 4/4 (100%) single WBCs amplified. An average yield of 578 ng (range of 227–1190 ng) was obtained from the single cell WGA reactions. (B) Concentrations of the next-generation sequencing libraries as measured by PicoGreen. All of the NGS libraries passed QC with adequate yield except for two samples that failed to render any detectable amount of library DNA product (one PC3 and WBC replica samples) (23/25; 92% success rate). An average yield of 581 ng (range of 397–1216 ng) was obtained among the single cell NGS libraries. (C) Assessment of NGS read quality. > 99% of the NGS reads had an average PHRED score greater than Q30 pre-alignment, indicating high quality reads. An average of 17 million reads/sample (in each direction) were obtained. 99% of the reads mapped to the reference genome (hg38) with 79% of the reads mapping with a MAPQ score greater than 30. *Failed library preparation.
    Figure Legend Snippet: Whole Genome Amplification, library preparation quality control, read quality, output, and alignment quality. (A) DNA concentrations and total yield for each single cell WGA as measured by UV/Vis. Libraries were constructed from independent replicates of single cells: 8 from LNCaP, 8 from PC3, 5 from VCaP, and 4 from WBCs. Overall, we achieved an average 100% success rate during the single cell whole genome amplification procedure: 8/8 single cells (100%) successfully amplified for PC3 and LNCaP cancer cell lines, while 5/5 (100%) single VCaP cells and 4/4 (100%) single WBCs amplified. An average yield of 578 ng (range of 227–1190 ng) was obtained from the single cell WGA reactions. (B) Concentrations of the next-generation sequencing libraries as measured by PicoGreen. All of the NGS libraries passed QC with adequate yield except for two samples that failed to render any detectable amount of library DNA product (one PC3 and WBC replica samples) (23/25; 92% success rate). An average yield of 581 ng (range of 397–1216 ng) was obtained among the single cell NGS libraries. (C) Assessment of NGS read quality. > 99% of the NGS reads had an average PHRED score greater than Q30 pre-alignment, indicating high quality reads. An average of 17 million reads/sample (in each direction) were obtained. 99% of the reads mapped to the reference genome (hg38) with 79% of the reads mapping with a MAPQ score greater than 30. *Failed library preparation.

    Techniques Used: Whole Genome Amplification, Construct, Amplification, Next-Generation Sequencing

    22) Product Images from "Genetic and epigenetic variation in 5S ribosomal RNA genes reveals genome dynamics in Arabidopsis thaliana"

    Article Title: Genetic and epigenetic variation in 5S ribosomal RNA genes reveals genome dynamics in Arabidopsis thaliana

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky163

    Variations of 5S rDNA copy number and genomic position. ( A ) (Top) Linkage mapping of the abundance of the T to C single nucleotide polymorphism at position 123 estimated by NGS in 393 individuals of the MAGIC population. (Bottom) Boxplots of the estimated founder ecotype effect by multiple imputation using R/happy at the major quantitative trait locus from the top panel in chromosome 5 and chromosome 3. ( B ) Number of total 5S rRNA genes and genes with chromosome 4 or 5 specific T-stretch signatures as determined by in silico analysis of La-0 and L er Illumina sequencing datasets. ( C ) FISH using LNA-DNA mixmer probes specific for chromosome 4 (red) and chromosome 5 (green) on pachytene spreads of Col-0 and L er . DNA is counterstained by DAPI (in grey). Arrow indicates weak cross hybridization of the chromosome 4 probe to the 5S rRNA gene copies from chromosome 3. Arrowhead indicates the 5S locus on the long arm of bivalent 3 in L er . The scale bar presents 5 μM. ( D ) FISH with 5S (red) and 45S (green) rDNA probes on metaphase I bivalents of Col-0, L er and the ago4 mutant alleles, ago4-2 (Col-0 background) and ago4-1 (L er background ) . The scale bar presents 5 μM. ( E ) FISH on ago4-2 and ago4-1 pachytene spreads with LNA-DNA mixmer probes as in (C). Arrow indicates weak cross hybridization of the chromosome 4 probe with the 5S rRNA gene copies of chromosome 3 in ago4-2 . Arrowhead designates the additional locus (green) on the long arm of bivalent 3 in ago4-2 . The locus on the long arm of bivalent 3 in L er is lost in ago4-1 . The scale bar presents 5 μM. ( F ) Relative 5S rDNA copy number for each locus in Col-0, ago4-2 , L er and ago4-1 determined by qPCR in three biological replicates. Copy numbers in Col-0 are set to 1 for each chromosome. * P
    Figure Legend Snippet: Variations of 5S rDNA copy number and genomic position. ( A ) (Top) Linkage mapping of the abundance of the T to C single nucleotide polymorphism at position 123 estimated by NGS in 393 individuals of the MAGIC population. (Bottom) Boxplots of the estimated founder ecotype effect by multiple imputation using R/happy at the major quantitative trait locus from the top panel in chromosome 5 and chromosome 3. ( B ) Number of total 5S rRNA genes and genes with chromosome 4 or 5 specific T-stretch signatures as determined by in silico analysis of La-0 and L er Illumina sequencing datasets. ( C ) FISH using LNA-DNA mixmer probes specific for chromosome 4 (red) and chromosome 5 (green) on pachytene spreads of Col-0 and L er . DNA is counterstained by DAPI (in grey). Arrow indicates weak cross hybridization of the chromosome 4 probe to the 5S rRNA gene copies from chromosome 3. Arrowhead indicates the 5S locus on the long arm of bivalent 3 in L er . The scale bar presents 5 μM. ( D ) FISH with 5S (red) and 45S (green) rDNA probes on metaphase I bivalents of Col-0, L er and the ago4 mutant alleles, ago4-2 (Col-0 background) and ago4-1 (L er background ) . The scale bar presents 5 μM. ( E ) FISH on ago4-2 and ago4-1 pachytene spreads with LNA-DNA mixmer probes as in (C). Arrow indicates weak cross hybridization of the chromosome 4 probe with the 5S rRNA gene copies of chromosome 3 in ago4-2 . Arrowhead designates the additional locus (green) on the long arm of bivalent 3 in ago4-2 . The locus on the long arm of bivalent 3 in L er is lost in ago4-1 . The scale bar presents 5 μM. ( F ) Relative 5S rDNA copy number for each locus in Col-0, ago4-2 , L er and ago4-1 determined by qPCR in three biological replicates. Copy numbers in Col-0 are set to 1 for each chromosome. * P

    Techniques Used: Next-Generation Sequencing, In Silico, Sequencing, Fluorescence In Situ Hybridization, Hybridization, Mutagenesis, Real-time Polymerase Chain Reaction

    23) Product Images from "Comprehensive nucleosome mapping of the human genome in cancer progression"

    Article Title: Comprehensive nucleosome mapping of the human genome in cancer progression

    Journal: Oncotarget

    doi: 10.18632/oncotarget.6811

    The newly developed mTSS-Capture method combined with paired-end sequencing maps genome-wide nucleosome distribution in primary patient samples and identifies bona fide nucleosome characteristics, concordant with other human nucleosome mapping studies A . Work-flow of the mTSS-seq method. Following MNase digestion using a titration of MNase, populations of mononucleosomally protected DNA and subnucleosomal fragments are isolated, and prepared as libraries for Illumina sequencing. Solution-based sequence capture is performed using biotinylated oligos, enabling the enrichment of fragments within 2kb of each transcription start site in the human genome. Paired-end 50bp sequencing was then performed on each index. B . Alignment of the mTSS-seq midpoints to the human genome using the UCSC genome browser for LAC patient #4137 Normal tissue is shown for chr11, hg19 ( http://genome.ucsc.edu ). Zooming in twice at 100X allows for further visualization of the sequence capture oligos surrounding the TSS in a 500kb and a 5kb region showing the ATM locus. C . Averaged, normalized reads per million (y-axis) from mTSS-seq plotted as fragments (gray) and midpoints (black), centered on and surrounding 2kb of the TSS for ~22,000 open reading frames in hg19 (x-axis). DNase I-hypersensitivity (GSM736580; green) and RNA polymerase II from ChIP-seq (GSM935299; blue) data from A549 cells are shown. (D) LAC patient 4137 Normal nucleosomal midpoints (blue track) were plotted in the UCSC genome browser against the published human lymphocyte nucleosome distribution maps by Gaffney et. al. (green track) for the ZNF451 and CCDC97 loci. Sequence capture oligos and corresponding RefSeq gene models are shown for each locus. Correlations are shown for ZNF451 and CCDC87, respectively.
    Figure Legend Snippet: The newly developed mTSS-Capture method combined with paired-end sequencing maps genome-wide nucleosome distribution in primary patient samples and identifies bona fide nucleosome characteristics, concordant with other human nucleosome mapping studies A . Work-flow of the mTSS-seq method. Following MNase digestion using a titration of MNase, populations of mononucleosomally protected DNA and subnucleosomal fragments are isolated, and prepared as libraries for Illumina sequencing. Solution-based sequence capture is performed using biotinylated oligos, enabling the enrichment of fragments within 2kb of each transcription start site in the human genome. Paired-end 50bp sequencing was then performed on each index. B . Alignment of the mTSS-seq midpoints to the human genome using the UCSC genome browser for LAC patient #4137 Normal tissue is shown for chr11, hg19 ( http://genome.ucsc.edu ). Zooming in twice at 100X allows for further visualization of the sequence capture oligos surrounding the TSS in a 500kb and a 5kb region showing the ATM locus. C . Averaged, normalized reads per million (y-axis) from mTSS-seq plotted as fragments (gray) and midpoints (black), centered on and surrounding 2kb of the TSS for ~22,000 open reading frames in hg19 (x-axis). DNase I-hypersensitivity (GSM736580; green) and RNA polymerase II from ChIP-seq (GSM935299; blue) data from A549 cells are shown. (D) LAC patient 4137 Normal nucleosomal midpoints (blue track) were plotted in the UCSC genome browser against the published human lymphocyte nucleosome distribution maps by Gaffney et. al. (green track) for the ZNF451 and CCDC97 loci. Sequence capture oligos and corresponding RefSeq gene models are shown for each locus. Correlations are shown for ZNF451 and CCDC87, respectively.

    Techniques Used: Sequencing, Genome Wide, Flow Cytometry, Titration, Isolation, Chromatin Immunoprecipitation

    24) Product Images from "Genetic and epigenetic variation in 5S ribosomal RNA genes reveals genome dynamics in Arabidopsis thaliana"

    Article Title: Genetic and epigenetic variation in 5S ribosomal RNA genes reveals genome dynamics in Arabidopsis thaliana

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky163

    Variations of 5S rDNA copy number and genomic position. ( A ) (Top) Linkage mapping of the abundance of the T to C single nucleotide polymorphism at position 123 estimated by NGS in 393 individuals of the MAGIC population. (Bottom) Boxplots of the estimated founder ecotype effect by multiple imputation using R/happy at the major quantitative trait locus from the top panel in chromosome 5 and chromosome 3. ( B ) Number of total 5S rRNA genes and genes with chromosome 4 or 5 specific T-stretch signatures as determined by in silico analysis of La-0 and L er Illumina sequencing datasets. ( C ) FISH using LNA-DNA mixmer probes specific for chromosome 4 (red) and chromosome 5 (green) on pachytene spreads of Col-0 and L er . DNA is counterstained by DAPI (in grey). Arrow indicates weak cross hybridization of the chromosome 4 probe to the 5S rRNA gene copies from chromosome 3. Arrowhead indicates the 5S locus on the long arm of bivalent 3 in L er . The scale bar presents 5 μM. ( D ) FISH with 5S (red) and 45S (green) rDNA probes on metaphase I bivalents of Col-0, L er and the ago4 mutant alleles, ago4-2 (Col-0 background) and ago4-1 (L er background ) . The scale bar presents 5 μM. ( E ) FISH on ago4-2 and ago4-1 pachytene spreads with LNA-DNA mixmer probes as in (C). Arrow indicates weak cross hybridization of the chromosome 4 probe with the 5S rRNA gene copies of chromosome 3 in ago4-2 . Arrowhead designates the additional locus (green) on the long arm of bivalent 3 in ago4-2 . The locus on the long arm of bivalent 3 in L er is lost in ago4-1 . The scale bar presents 5 μM. ( F ) Relative 5S rDNA copy number for each locus in Col-0, ago4-2 , L er and ago4-1 determined by qPCR in three biological replicates. Copy numbers in Col-0 are set to 1 for each chromosome. * P
    Figure Legend Snippet: Variations of 5S rDNA copy number and genomic position. ( A ) (Top) Linkage mapping of the abundance of the T to C single nucleotide polymorphism at position 123 estimated by NGS in 393 individuals of the MAGIC population. (Bottom) Boxplots of the estimated founder ecotype effect by multiple imputation using R/happy at the major quantitative trait locus from the top panel in chromosome 5 and chromosome 3. ( B ) Number of total 5S rRNA genes and genes with chromosome 4 or 5 specific T-stretch signatures as determined by in silico analysis of La-0 and L er Illumina sequencing datasets. ( C ) FISH using LNA-DNA mixmer probes specific for chromosome 4 (red) and chromosome 5 (green) on pachytene spreads of Col-0 and L er . DNA is counterstained by DAPI (in grey). Arrow indicates weak cross hybridization of the chromosome 4 probe to the 5S rRNA gene copies from chromosome 3. Arrowhead indicates the 5S locus on the long arm of bivalent 3 in L er . The scale bar presents 5 μM. ( D ) FISH with 5S (red) and 45S (green) rDNA probes on metaphase I bivalents of Col-0, L er and the ago4 mutant alleles, ago4-2 (Col-0 background) and ago4-1 (L er background ) . The scale bar presents 5 μM. ( E ) FISH on ago4-2 and ago4-1 pachytene spreads with LNA-DNA mixmer probes as in (C). Arrow indicates weak cross hybridization of the chromosome 4 probe with the 5S rRNA gene copies of chromosome 3 in ago4-2 . Arrowhead designates the additional locus (green) on the long arm of bivalent 3 in ago4-2 . The locus on the long arm of bivalent 3 in L er is lost in ago4-1 . The scale bar presents 5 μM. ( F ) Relative 5S rDNA copy number for each locus in Col-0, ago4-2 , L er and ago4-1 determined by qPCR in three biological replicates. Copy numbers in Col-0 are set to 1 for each chromosome. * P

    Techniques Used: Next-Generation Sequencing, In Silico, Sequencing, Fluorescence In Situ Hybridization, Hybridization, Mutagenesis, Real-time Polymerase Chain Reaction

    25) Product Images from "Ultrasensitive and high-efficiency screen of de novo low-frequency mutations by o2n-seq"

    Article Title: Ultrasensitive and high-efficiency screen of de novo low-frequency mutations by o2n-seq

    Journal: Nature Communications

    doi: 10.1038/ncomms15335

    Overview of o2n-seq. ( a ) O2n-seq workflow. gDNA is sheared into fragments shorter than half the length of the sequencing read (for example, if we sequence reads with PE 125, the length of fragments should shorter than 125 bp) and ligated with Y-shaped adaptors (blue and orange lines) with candidate nicking sites (such as dUTP), then denatured into single-stranded DNA molecules that are circularized using single-stranded DNA ligase. After eliminating linear DNA using DNA exonucleases, the circularized single-stranded DNA is employed for second-strand synthesis. Next, the double-stranded circular DNA is nicked with the USER enzyme and subjected to strand displacement amplification. The purified, strand displacement-amplified DNA can be used to prepare a standard NGS library. The arrows represent the direction of DNA (5′→3′). ( b ) Remove errors. If a variant (red star) is support by only one DNA copy, an error must have occurred at the site, and this type of site is discarded in the following data analysis procedure. However, if a variant is supported by both DNA copies, it is treated as a true variation. ( c ) Using a PE sequencing strategy to sequence o2n-seq reads. Read 1 and Read 2 are sequencing reads of one PE read.
    Figure Legend Snippet: Overview of o2n-seq. ( a ) O2n-seq workflow. gDNA is sheared into fragments shorter than half the length of the sequencing read (for example, if we sequence reads with PE 125, the length of fragments should shorter than 125 bp) and ligated with Y-shaped adaptors (blue and orange lines) with candidate nicking sites (such as dUTP), then denatured into single-stranded DNA molecules that are circularized using single-stranded DNA ligase. After eliminating linear DNA using DNA exonucleases, the circularized single-stranded DNA is employed for second-strand synthesis. Next, the double-stranded circular DNA is nicked with the USER enzyme and subjected to strand displacement amplification. The purified, strand displacement-amplified DNA can be used to prepare a standard NGS library. The arrows represent the direction of DNA (5′→3′). ( b ) Remove errors. If a variant (red star) is support by only one DNA copy, an error must have occurred at the site, and this type of site is discarded in the following data analysis procedure. However, if a variant is supported by both DNA copies, it is treated as a true variation. ( c ) Using a PE sequencing strategy to sequence o2n-seq reads. Read 1 and Read 2 are sequencing reads of one PE read.

    Techniques Used: Sequencing, Amplification, Purification, Next-Generation Sequencing, Variant Assay

    Related Articles

    Amplification:

    Article Title: The contribution of pathogenic variants in breast cancer susceptibility genes to familial breast cancer risk
    Article Snippet: Library preparation was performed using the NEBNext Ultra DNA Library Prep kit (E7370L), and NEB Dual indexed adapters. .. Fragmented DNA (mean fragment size 300 ± 20 bp) was end repaired, adapter ligated, and size selected using Ampure XP /SPRI (A63881) and was PCR amplified for eight cycles.

    Article Title: DNA copy number changes define spatial patterns of heterogeneity in colorectal cancer
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    Article Title: Unliganded Progesterone Receptor Governs Estrogen Receptor Gene Expression by Regulating DNA Methylation in Breast Cancer Cells
    Article Snippet: .. Fragmented DNA was immunoprecipitated with antibody against 5mC as described above, and the amplified library was prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina (E7370L) following the manufacturer’s instructions. .. Amplified libraries were sequenced, and reads were aligned with BowTie v1.1.2 using the reference human genome Version 19 (hg19), as previously described [ ].

    Article Title: Astrocytes locally translate transcripts in their peripheral processes
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    DNA Ligation:

    Article Title: Characterization of the Chloroplast Genome of Trentepohlia odorata (Trentepohliales, Chlorophyta), and Discussion of its Taxonomy
    Article Snippet: Chloroplast Genome Assembly and Annotation A paired-end Illumina sequencing library was prepared from total DNA using the NEBNext Ultra DNA Library Prep Kit (E7370S). .. A 1D genomic DNA by ligation (SQK-LSK108) kit was used to construct a long-reads library according to the manufacturer’s instructions.

    Synthesized:

    Article Title: A new approach for detecting adventitious viruses shows Sf-rhabdovirus-negative Sf-RVN cells are suitable for safe biologicals production
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    Methylated DNA Immunoprecipitation Sequencing:

    Article Title: Unliganded Progesterone Receptor Governs Estrogen Receptor Gene Expression by Regulating DNA Methylation in Breast Cancer Cells
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    Construct:

    Article Title: Draft Genome Sequence of Pseudomonas syringae pv. persicae NCPPB 2254
    Article Snippet: .. Three DNA libraries were constructed from the extracted genomic DNA using a NEB E7370L DNA library preparation kit for Illumina, with average insert sizes of 330 bp, 460 bp, and 600 bp, respectively. .. The sequencing was performed on an Illumina MiSeq platform using a MiSeq reagent kit v3, 600 cycles.

    Article Title: An efficient pipeline to generate data for studies in plastid population genomics and phylogeography 1
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    Real-time Polymerase Chain Reaction:

    Article Title: Unliganded Progesterone Receptor Governs Estrogen Receptor Gene Expression by Regulating DNA Methylation in Breast Cancer Cells
    Article Snippet: The immunocomplexes were recovered using 8 μL Dynabeads (M-280; Thermofisher, Waltham, MA, USA), and the pull-down products were detected by quantitative-PCR. .. Fragmented DNA was immunoprecipitated with antibody against 5mC as described above, and the amplified library was prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina (E7370L) following the manufacturer’s instructions.

    Article Title: A new approach for detecting adventitious viruses shows Sf-rhabdovirus-negative Sf-RVN cells are suitable for safe biologicals production
    Article Snippet: For genome sequencing, a DNA library was prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina (E7370, New England Biolabs) following the manufacturer’s recommendations. .. Adapter-ligated DNAs were indexed and enriched by limited cycle PCR, quantified by real-time PCR, and multiplexed by equal molar mass.

    Incubation:

    Article Title: Synthetic essentiality of chromatin remodeling factor CHD1 in PTEN deficient cancer
    Article Snippet: Solubilized chromatin was then incubated overnight with their respective antibody-Dynabead (Life Technologies) mixture (CHD1 antibody: Bethyl, #A301-218A; H3K4me3 antibody: Abcam #ab8580). .. Libraries were prepared using NEBNext Ultra DNA Library kit (E7370).

    Article Title: H3K9 methyltransferases and demethylases control lung tumor-propagating cells and lung cancer progression
    Article Snippet: Fragmented chromatin was diluted in immunoprecipitation (IP) buffer (20 mM Tris-HCl, pH 8.1, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100) and incubated overnight at 4 °C with Protein G magnetic beads (Dynabeads: Life Technologies) that had been pre-incubated with H3K9me2 (Abcam, ab1220, 62.5 ng/µl) or H3K9me1 (Abcam, ab8896, 62.5 ng/µl) antibodies. .. One to ten nanograms of DNA were used for the library construction using NEBNext Ultra DNA Library Prep Kit (NEB E7370).

    Formalin-fixed Paraffin-Embedded:

    Article Title: DNA copy number changes define spatial patterns of heterogeneity in colorectal cancer
    Article Snippet: .. DNA (10 ng) from FFPE embedded tumour tissue was prepared using the Ion AmpliSeq Library Kit 2.0 (Life technologies) for the CRC panel, followed by library preparation using the NEB Next Ultra DNA Library Prep kit (end repair, A-tailing, adaptor ligation and amplification; NEB, E7370S) and NEB Next Multiplex Oligos provided for Illumina (NEB, E7335S). ..

    Genome Wide:

    Article Title: Unliganded Progesterone Receptor Governs Estrogen Receptor Gene Expression by Regulating DNA Methylation in Breast Cancer Cells
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    Ligation:

    Article Title: DNA copy number changes define spatial patterns of heterogeneity in colorectal cancer
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    Molecular Weight:

    Article Title: The contribution of pathogenic variants in breast cancer susceptibility genes to familial breast cancer risk
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    Cell Culture:

    Article Title: A new approach for detecting adventitious viruses shows Sf-rhabdovirus-negative Sf-RVN cells are suitable for safe biologicals production
    Article Snippet: Paragraph title: Cell culture, DNA and RNA extraction, MPS ... For genome sequencing, a DNA library was prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina (E7370, New England Biolabs) following the manufacturer’s recommendations.

    Generated:

    Article Title: Draft Genome Sequence of Pseudomonas syringae pv. persicae NCPPB 2254
    Article Snippet: Three DNA libraries were constructed from the extracted genomic DNA using a NEB E7370L DNA library preparation kit for Illumina, with average insert sizes of 330 bp, 460 bp, and 600 bp, respectively. .. A total of 2,931,855 × 2 × 301 bp generated reads were subjected to the skewer ( ) program (version 0.1.123) with a quality threshold of 9 and an error threshold of 0.25 for adapter trimming, and then de novo assembled using the ABySS program ( ) (version 1.5.2) with a k-mer size of 128.

    Article Title: An efficient pipeline to generate data for studies in plastid population genomics and phylogeography 1
    Article Snippet: Library preparation was conducted using a NEBNext Ultra DNA Library Prep Kit (E7370) with NEBNext Multiplex Oligos (E7600; New England Biolabs, Ipswich, Massachusetts, USA). .. DNA was sequenced on an Illumina HiSeq 2500 Sequencer (Illumina), with 100-bp paired-end reads generated for all but six samples, which had 100-bp single-end reads (Massively Parallel Sequencing Shared Resource Facility, Oregon Health and Science University, Portland, Oregon, USA).

    Article Title: Unliganded Progesterone Receptor Governs Estrogen Receptor Gene Expression by Regulating DNA Methylation in Breast Cancer Cells
    Article Snippet: Fragmented DNA was immunoprecipitated with antibody against 5mC as described above, and the amplified library was prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina (E7370L) following the manufacturer’s instructions. .. The 5mC and CpG heat maps were generated using DeepTools (Version 2.2.0) [ ] and BEDtools (Version v2.24.0) [ ], and the matrix underlying the heatmaps was used to generate the 5mC and CpG average profiles.

    Article Title: Astrocytes locally translate transcripts in their peripheral processes
    Article Snippet: .. Illumina sequencing libraries were generated from 600 ng of amplified cDNA, after shearing to ∼200 bp using a Covaris sonicator (New England Biolabs Next Ultra DNA Library Prep Kit for Illumina, #E7370S). .. Libraries were sequenced on an Illumina HiSEq.

    Sequencing:

    Article Title: Significant heterogeneity in Wolbachia copy number within and between populations of Onchocerca volvulus
    Article Snippet: .. Briefly, genomic DNA for individual worms was sheared to an average fragment length of 400 bp, and barcoded Illumina sequencing libraries for each individual worm were prepared using the NEBNext® Ultra™ DNA Library Prep kit for Illumina (E7370L, New England Biolabs, Inc. Ipswich, USA). .. The barcoded single worm libraries for up to 16 worms were pooled per Ilumina HiSeq lane and paired-end sequenced (Illumina Inc., San Diego, USA).

    Article Title: Synthetic essentiality of chromatin remodeling factor CHD1 in PTEN deficient cancer
    Article Snippet: Libraries were prepared using NEBNext Ultra DNA Library kit (E7370). .. Sequencing was performed using an Illumina HiSeq 2500 instrument to generate a dataset .

    Article Title: Intrinsic cooperativity potentiates parallel cis-regulatory evolution
    Article Snippet: .. Genomic DNA was prepared for sequencing using the NEBNext Ultra DNA Prep Kit for Illumina E7370 (New England BioLabs). .. All three isolates of each of the four hybrids were prepared for mRNA sequencing.

    Article Title: The contribution of pathogenic variants in breast cancer susceptibility genes to familial breast cancer risk
    Article Snippet: Target selection and library preparation The target region for sequencing of the candidate breast cancer susceptibility genes were selected based on coding exons. .. Library preparation was performed using the NEBNext Ultra DNA Library Prep kit (E7370L), and NEB Dual indexed adapters.

    Article Title: H3K9 methyltransferases and demethylases control lung tumor-propagating cells and lung cancer progression
    Article Snippet: One to ten nanograms of DNA were used for the library construction using NEBNext Ultra DNA Library Prep Kit (NEB E7370). .. Sequencing was performed on a NextSeq (Illumina) for 38 nucleotides from paired ends according to the manufacturer’s instructions.

    Article Title: DNA copy number changes define spatial patterns of heterogeneity in colorectal cancer
    Article Snippet: MiSeq (Illumina) re-sequencing We used a MiSeq device (Illumina) as a second platform for validating sequencing results for patients D11 and D30 using the same custom CRC panel. .. DNA (10 ng) from FFPE embedded tumour tissue was prepared using the Ion AmpliSeq Library Kit 2.0 (Life technologies) for the CRC panel, followed by library preparation using the NEB Next Ultra DNA Library Prep kit (end repair, A-tailing, adaptor ligation and amplification; NEB, E7370S) and NEB Next Multiplex Oligos provided for Illumina (NEB, E7335S).

    Article Title: Draft Genome Sequence of Pseudomonas syringae pv. persicae NCPPB 2254
    Article Snippet: Here, we briefly describe the genome sequencing of P. syringae pv. persicae strain NCPPB 2254. .. Three DNA libraries were constructed from the extracted genomic DNA using a NEB E7370L DNA library preparation kit for Illumina, with average insert sizes of 330 bp, 460 bp, and 600 bp, respectively.

    Article Title: An efficient pipeline to generate data for studies in plastid population genomics and phylogeography 1
    Article Snippet: Library preparation was conducted using a NEBNext Ultra DNA Library Prep Kit (E7370) with NEBNext Multiplex Oligos (E7600; New England Biolabs, Ipswich, Massachusetts, USA). .. DNA was sequenced on an Illumina HiSeq 2500 Sequencer (Illumina), with 100-bp paired-end reads generated for all but six samples, which had 100-bp single-end reads (Massively Parallel Sequencing Shared Resource Facility, Oregon Health and Science University, Portland, Oregon, USA).

    Article Title: Astrocytes locally translate transcripts in their peripheral processes
    Article Snippet: .. Illumina sequencing libraries were generated from 600 ng of amplified cDNA, after shearing to ∼200 bp using a Covaris sonicator (New England Biolabs Next Ultra DNA Library Prep Kit for Illumina, #E7370S). .. Libraries were sequenced on an Illumina HiSEq.

    Article Title: Characterization of the Chloroplast Genome of Trentepohlia odorata (Trentepohliales, Chlorophyta), and Discussion of its Taxonomy
    Article Snippet: .. Chloroplast Genome Assembly and Annotation A paired-end Illumina sequencing library was prepared from total DNA using the NEBNext Ultra DNA Library Prep Kit (E7370S). .. The libraries were sequenced using an Illumina NovaSeq 6000 with 150 bp insertion fragments (Illumina, San Diego, CA, USA).

    Sonication:

    Article Title: H3K9 methyltransferases and demethylases control lung tumor-propagating cells and lung cancer progression
    Article Snippet: ChIP and ChIP-seq Treated cell lines were crosslinked with 1% formaldehyde in PBS for 10 min at room temperature (RT), washed in 5 mg ml−1 bovine serum albumin in PBS and then in just cold PBS, re-suspended in lysis buffer (50 mM Tris-HCl, pH 8.1, 10 mM EDTA, 1% sodium dodecyl sulfate (SDS), 1× complete protease inhibitors (Roche)) and sonicated with the Covaris M220 sonicator to obtain chromatin fragment lengths of 200–500 bp judged by Bioanalyzer DNA High Sensitivity Kit (Agilent). .. One to ten nanograms of DNA were used for the library construction using NEBNext Ultra DNA Library Prep Kit (NEB E7370).

    Article Title: Unliganded Progesterone Receptor Governs Estrogen Receptor Gene Expression by Regulating DNA Methylation in Breast Cancer Cells
    Article Snippet: Sonicated DNA was denatured and then immunoprecipitated using an antibody against 5mC (Eurogentec; #BI-MECY-1000) or mouse IgG antibody, as previously described [ ]. .. Fragmented DNA was immunoprecipitated with antibody against 5mC as described above, and the amplified library was prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina (E7370L) following the manufacturer’s instructions.

    Magnetic Cell Separation:

    Article Title: H3K9 methyltransferases and demethylases control lung tumor-propagating cells and lung cancer progression
    Article Snippet: One to ten nanograms of DNA were used for the library construction using NEBNext Ultra DNA Library Prep Kit (NEB E7370). .. H3K27Ac-modified regions were identified using MACS (version 1.4.2) as previously described , with a P value cutoff of 1E−5 and with default values for other parameters.

    DNA Extraction:

    Article Title: An efficient pipeline to generate data for studies in plastid population genomics and phylogeography 1
    Article Snippet: After DNA extraction, DNA concentration was quantified on a Qubit 3.0 fluorometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and pooled by population in an equimolar fashion (20 samples per PL). .. Library preparation was conducted using a NEBNext Ultra DNA Library Prep Kit (E7370) with NEBNext Multiplex Oligos (E7600; New England Biolabs, Ipswich, Massachusetts, USA).

    RNA Sequencing Assay:

    Article Title: Astrocytes locally translate transcripts in their peripheral processes
    Article Snippet: Double-stranded cDNA was prepared from 2 ng of RNA using the Nugen Ovation RNA-seq System V2. .. Illumina sequencing libraries were generated from 600 ng of amplified cDNA, after shearing to ∼200 bp using a Covaris sonicator (New England Biolabs Next Ultra DNA Library Prep Kit for Illumina, #E7370S).

    Magnetic Beads:

    Article Title: H3K9 methyltransferases and demethylases control lung tumor-propagating cells and lung cancer progression
    Article Snippet: Fragmented chromatin was diluted in immunoprecipitation (IP) buffer (20 mM Tris-HCl, pH 8.1, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100) and incubated overnight at 4 °C with Protein G magnetic beads (Dynabeads: Life Technologies) that had been pre-incubated with H3K9me2 (Abcam, ab1220, 62.5 ng/µl) or H3K9me1 (Abcam, ab8896, 62.5 ng/µl) antibodies. .. One to ten nanograms of DNA were used for the library construction using NEBNext Ultra DNA Library Prep Kit (NEB E7370).

    Multiplex Assay:

    Article Title: DNA copy number changes define spatial patterns of heterogeneity in colorectal cancer
    Article Snippet: .. DNA (10 ng) from FFPE embedded tumour tissue was prepared using the Ion AmpliSeq Library Kit 2.0 (Life technologies) for the CRC panel, followed by library preparation using the NEB Next Ultra DNA Library Prep kit (end repair, A-tailing, adaptor ligation and amplification; NEB, E7370S) and NEB Next Multiplex Oligos provided for Illumina (NEB, E7335S). ..

    Article Title: An efficient pipeline to generate data for studies in plastid population genomics and phylogeography 1
    Article Snippet: .. Library preparation was conducted using a NEBNext Ultra DNA Library Prep Kit (E7370) with NEBNext Multiplex Oligos (E7600; New England Biolabs, Ipswich, Massachusetts, USA). .. SSLs were constructed for at least one individual from each population.

    Purification:

    Article Title: Synthetic essentiality of chromatin remodeling factor CHD1 in PTEN deficient cancer
    Article Snippet: Eluted DNA was purified using AMPure beads (Beckman-Coulter). .. Libraries were prepared using NEBNext Ultra DNA Library kit (E7370).

    Article Title: H3K9 methyltransferases and demethylases control lung tumor-propagating cells and lung cancer progression
    Article Snippet: Immunoprecipitated (or no IP input) DNA was treated with RNase A and proteinase K on the beads, recovered in 1% SDS and 0.1 M NaHCO3 over a period of 5 h at 65 °C, and column purified with DNA clean and concentrator-25 (Zymo Research). .. One to ten nanograms of DNA were used for the library construction using NEBNext Ultra DNA Library Prep Kit (NEB E7370).

    Polymerase Chain Reaction:

    Article Title: Genome-wide analysis of replication timing by next-generation sequencing with E/L Repli-seq
    Article Snippet: Add 90μL of resuspended AMPure XP beads to the 100μL PCR reaction. .. CRITICAL STEP If you start from poorly fragmented DNA and you are sure you need to perform size selection at this moment, refer to NEB manual E7370 and optimize the volume of AMPure XP beads to use.

    Article Title: The contribution of pathogenic variants in breast cancer susceptibility genes to familial breast cancer risk
    Article Snippet: Library preparation was performed using the NEBNext Ultra DNA Library Prep kit (E7370L), and NEB Dual indexed adapters. .. Fragmented DNA (mean fragment size 300 ± 20 bp) was end repaired, adapter ligated, and size selected using Ampure XP /SPRI (A63881) and was PCR amplified for eight cycles.

    Article Title: A new approach for detecting adventitious viruses shows Sf-rhabdovirus-negative Sf-RVN cells are suitable for safe biologicals production
    Article Snippet: For genome sequencing, a DNA library was prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina (E7370, New England Biolabs) following the manufacturer’s recommendations. .. Adapter-ligated DNAs were indexed and enriched by limited cycle PCR, quantified by real-time PCR, and multiplexed by equal molar mass.

    Immunoprecipitation:

    Article Title: H3K9 methyltransferases and demethylases control lung tumor-propagating cells and lung cancer progression
    Article Snippet: Immunoprecipitated (or no IP input) DNA was treated with RNase A and proteinase K on the beads, recovered in 1% SDS and 0.1 M NaHCO3 over a period of 5 h at 65 °C, and column purified with DNA clean and concentrator-25 (Zymo Research). .. One to ten nanograms of DNA were used for the library construction using NEBNext Ultra DNA Library Prep Kit (NEB E7370).

    Article Title: Unliganded Progesterone Receptor Governs Estrogen Receptor Gene Expression by Regulating DNA Methylation in Breast Cancer Cells
    Article Snippet: .. Fragmented DNA was immunoprecipitated with antibody against 5mC as described above, and the amplified library was prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina (E7370L) following the manufacturer’s instructions. .. Amplified libraries were sequenced, and reads were aligned with BowTie v1.1.2 using the reference human genome Version 19 (hg19), as previously described [ ].

    Chromatin Immunoprecipitation:

    Article Title: H3K9 methyltransferases and demethylases control lung tumor-propagating cells and lung cancer progression
    Article Snippet: Paragraph title: ChIP and ChIP-seq ... One to ten nanograms of DNA were used for the library construction using NEBNext Ultra DNA Library Prep Kit (NEB E7370).

    Article Title: DNA methylation heterogeneity defines a disease spectrum in Ewing sarcoma
    Article Snippet: .. Library preparation for ChIP DNA and input control DNA was performed using the NEBNext Ultra kit (New England Biolabs, E7370S/L) following the manufacturer’s instructions. .. Quality control for the final libraries was done by measuring the DNA concentration with the Qubit dsDNA HS assay (Life Technologies, ) on Qubit 2.0 Fluorometer (Life Technologies, ) and by determining library fragment sizes with the Experion DNA 1K Analysis kit (Bio-Rad, 700-7107) on the Experion Automated Electrophoresis Station (Bio-Rad, 701-7000).

    Software:

    Article Title: Significant heterogeneity in Wolbachia copy number within and between populations of Onchocerca volvulus
    Article Snippet: Briefly, genomic DNA for individual worms was sheared to an average fragment length of 400 bp, and barcoded Illumina sequencing libraries for each individual worm were prepared using the NEBNext® Ultra™ DNA Library Prep kit for Illumina (E7370L, New England Biolabs, Inc. Ipswich, USA). .. After standard trimming to remove adaptor and barcode sequences, and filtering for quality using Trimmomatic [ ], BWA-MEM software [ ] was used to map reads to the Wolbachia nuclear and mitochondrial reference genomes of O. volvulus .

    Electrophoresis:

    Article Title: DNA methylation heterogeneity defines a disease spectrum in Ewing sarcoma
    Article Snippet: Library preparation for ChIP DNA and input control DNA was performed using the NEBNext Ultra kit (New England Biolabs, E7370S/L) following the manufacturer’s instructions. .. Quality control for the final libraries was done by measuring the DNA concentration with the Qubit dsDNA HS assay (Life Technologies, ) on Qubit 2.0 Fluorometer (Life Technologies, ) and by determining library fragment sizes with the Experion DNA 1K Analysis kit (Bio-Rad, 700-7107) on the Experion Automated Electrophoresis Station (Bio-Rad, 701-7000).

    RNA Extraction:

    Article Title: A new approach for detecting adventitious viruses shows Sf-rhabdovirus-negative Sf-RVN cells are suitable for safe biologicals production
    Article Snippet: Paragraph title: Cell culture, DNA and RNA extraction, MPS ... For genome sequencing, a DNA library was prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina (E7370, New England Biolabs) following the manufacturer’s recommendations.

    Selection:

    Article Title: Genome-wide analysis of replication timing by next-generation sequencing with E/L Repli-seq
    Article Snippet: .. CRITICAL STEP If you start from poorly fragmented DNA and you are sure you need to perform size selection at this moment, refer to NEB manual E7370 and optimize the volume of AMPure XP beads to use. .. Briefly, save the supernatant from the first selection to remove larger fragments which bind to the beads, then add more\ AMPure XP beads to bind the fragments of target size and discard the supernatant this time, wash the beads with 80% ethanol as Steps 66–69 and elute the target size fragments from the beads as Step 70.

    Article Title: The contribution of pathogenic variants in breast cancer susceptibility genes to familial breast cancer risk
    Article Snippet: Paragraph title: Target selection and library preparation ... Library preparation was performed using the NEBNext Ultra DNA Library Prep kit (E7370L), and NEB Dual indexed adapters.

    Next-Generation Sequencing:

    Article Title: Significant heterogeneity in Wolbachia copy number within and between populations of Onchocerca volvulus
    Article Snippet: Paragraph title: Relative copy number estimation by next-generation sequencing ... Briefly, genomic DNA for individual worms was sheared to an average fragment length of 400 bp, and barcoded Illumina sequencing libraries for each individual worm were prepared using the NEBNext® Ultra™ DNA Library Prep kit for Illumina (E7370L, New England Biolabs, Inc. Ipswich, USA).

    DNA Methylation Assay:

    Article Title: Unliganded Progesterone Receptor Governs Estrogen Receptor Gene Expression by Regulating DNA Methylation in Breast Cancer Cells
    Article Snippet: Paragraph title: 4.6. DNA Methylation ... Fragmented DNA was immunoprecipitated with antibody against 5mC as described above, and the amplified library was prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina (E7370L) following the manufacturer’s instructions.

    Concentration Assay:

    Article Title: An efficient pipeline to generate data for studies in plastid population genomics and phylogeography 1
    Article Snippet: After DNA extraction, DNA concentration was quantified on a Qubit 3.0 fluorometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and pooled by population in an equimolar fashion (20 samples per PL). .. Library preparation was conducted using a NEBNext Ultra DNA Library Prep Kit (E7370) with NEBNext Multiplex Oligos (E7600; New England Biolabs, Ipswich, Massachusetts, USA).

    Article Title: DNA methylation heterogeneity defines a disease spectrum in Ewing sarcoma
    Article Snippet: Library preparation for ChIP DNA and input control DNA was performed using the NEBNext Ultra kit (New England Biolabs, E7370S/L) following the manufacturer’s instructions. .. Quality control for the final libraries was done by measuring the DNA concentration with the Qubit dsDNA HS assay (Life Technologies, ) on Qubit 2.0 Fluorometer (Life Technologies, ) and by determining library fragment sizes with the Experion DNA 1K Analysis kit (Bio-Rad, 700-7107) on the Experion Automated Electrophoresis Station (Bio-Rad, 701-7000).

    ChIP-sequencing:

    Article Title: Synthetic essentiality of chromatin remodeling factor CHD1 in PTEN deficient cancer
    Article Snippet: Paragraph title: Chromatin Immunoprecipitation Sequencing (ChIP-seq) ... Libraries were prepared using NEBNext Ultra DNA Library kit (E7370).

    Article Title: H3K9 methyltransferases and demethylases control lung tumor-propagating cells and lung cancer progression
    Article Snippet: Paragraph title: ChIP and ChIP-seq ... One to ten nanograms of DNA were used for the library construction using NEBNext Ultra DNA Library Prep Kit (NEB E7370).

    Article Title: DNA methylation heterogeneity defines a disease spectrum in Ewing sarcoma
    Article Snippet: Paragraph title: ChIP-seq ... Library preparation for ChIP DNA and input control DNA was performed using the NEBNext Ultra kit (New England Biolabs, E7370S/L) following the manufacturer’s instructions.

    High Throughput Screening Assay:

    Article Title: Characterization of the Chloroplast Genome of Trentepohlia odorata (Trentepohliales, Chlorophyta), and Discussion of its Taxonomy
    Article Snippet: Chloroplast Genome Assembly and Annotation A paired-end Illumina sequencing library was prepared from total DNA using the NEBNext Ultra DNA Library Prep Kit (E7370S). .. High-throughput sequencing data was sequentially analyzed by SOAPnuke v1.3.0 and SPAdes v3.10.0 [ , ].

    Lysis:

    Article Title: H3K9 methyltransferases and demethylases control lung tumor-propagating cells and lung cancer progression
    Article Snippet: ChIP and ChIP-seq Treated cell lines were crosslinked with 1% formaldehyde in PBS for 10 min at room temperature (RT), washed in 5 mg ml−1 bovine serum albumin in PBS and then in just cold PBS, re-suspended in lysis buffer (50 mM Tris-HCl, pH 8.1, 10 mM EDTA, 1% sodium dodecyl sulfate (SDS), 1× complete protease inhibitors (Roche)) and sonicated with the Covaris M220 sonicator to obtain chromatin fragment lengths of 200–500 bp judged by Bioanalyzer DNA High Sensitivity Kit (Agilent). .. One to ten nanograms of DNA were used for the library construction using NEBNext Ultra DNA Library Prep Kit (NEB E7370).

    Methylated DNA Immunoprecipitation:

    Article Title: Unliganded Progesterone Receptor Governs Estrogen Receptor Gene Expression by Regulating DNA Methylation in Breast Cancer Cells
    Article Snippet: For MeDIP-qPCR, genomic DNA was randomly sheared by sonication to generate fragments between 300 and 700 bp. .. Fragmented DNA was immunoprecipitated with antibody against 5mC as described above, and the amplified library was prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina (E7370L) following the manufacturer’s instructions.

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    New England Biolabs ultra dna library prep kit
    Analysis of <t>Illumina</t> sequence reads of a black molly ( Poecilia cf. sphenops ) whole fish <t>DNA</t> library before and after enrichment with MBD-Fc protein A paramagnetic beads. Shown is a concordance plot comparing relative abundances of microbial genera between the enriched and unenriched samples.
    Ultra Dna Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 176 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Analysis of Illumina sequence reads of a black molly ( Poecilia cf. sphenops ) whole fish DNA library before and after enrichment with MBD-Fc protein A paramagnetic beads. Shown is a concordance plot comparing relative abundances of microbial genera between the enriched and unenriched samples.

    Journal: PLoS ONE

    Article Title: A Method for Selectively Enriching Microbial DNA from Contaminating Vertebrate Host DNA

    doi: 10.1371/journal.pone.0076096

    Figure Lengend Snippet: Analysis of Illumina sequence reads of a black molly ( Poecilia cf. sphenops ) whole fish DNA library before and after enrichment with MBD-Fc protein A paramagnetic beads. Shown is a concordance plot comparing relative abundances of microbial genera between the enriched and unenriched samples.

    Article Snippet: Preparation of Next-generation Sequencing Libraries DNA extracted from saliva and leukocytes were used to generate SOLiD 4 libraries using NEBNext® Fast DNA Library Prep Set for Ion Torrent (NEB #E6270) with SOLiD 4 adaptors and primers; mixtures of DNA extracted form E. coli K-12 MG1655 and IMR-90 cells were used to generate Ion Torrent libraries using NEBNext Fast DNA Library Prep Set for Ion Torrent (NEB #E6270); DNA extracted from black molly was used to generate Illumina paired end sequencing libraries using NEBNext Ultra DNA Library Prep Kit and Multiplex Oligos for Illumina (NEB #E7370 and #E7335).

    Techniques: Sequencing, Fluorescence In Situ Hybridization

    MBD-Fc separates human DNA from human-malarial DNA mixtures. (A) Graph of the percentage of 75 bp Illumina reads mapping to the Plasmodium falciparum reference sequence in the mixture before enrichment (Unenriched), after enrichment (Enriched) and the bound fraction following wash and elution as described above (Bound). (B) Evenness of coverage analysis metrics show enriched malaria reads (Enriched Plasmodium ) in line with pure malaria DNA reads ( Plasmodium Only). Unlike the unenriched input sample (Human + Plasmodium ) showing 60% of the Plasmodium genome uncovered (zero depth), enriched sample (Enriched Plasmodium ) showed even coverage of the genome with no regions lacking coverage. The amount of Plasmodium DNA retained in the pellet (Bound Human) following wash and elution was insignificant. (C) GC-content and bias analysis. No base bias was detected in the enriched sample. Average GC content of the enriched sample (Enriched Plasmodium ) matches the pure malaria sample ( Plasmodium Only) and is very close to the theoretical GC coverage (Theoretical).

    Journal: PLoS ONE

    Article Title: A Method for Selectively Enriching Microbial DNA from Contaminating Vertebrate Host DNA

    doi: 10.1371/journal.pone.0076096

    Figure Lengend Snippet: MBD-Fc separates human DNA from human-malarial DNA mixtures. (A) Graph of the percentage of 75 bp Illumina reads mapping to the Plasmodium falciparum reference sequence in the mixture before enrichment (Unenriched), after enrichment (Enriched) and the bound fraction following wash and elution as described above (Bound). (B) Evenness of coverage analysis metrics show enriched malaria reads (Enriched Plasmodium ) in line with pure malaria DNA reads ( Plasmodium Only). Unlike the unenriched input sample (Human + Plasmodium ) showing 60% of the Plasmodium genome uncovered (zero depth), enriched sample (Enriched Plasmodium ) showed even coverage of the genome with no regions lacking coverage. The amount of Plasmodium DNA retained in the pellet (Bound Human) following wash and elution was insignificant. (C) GC-content and bias analysis. No base bias was detected in the enriched sample. Average GC content of the enriched sample (Enriched Plasmodium ) matches the pure malaria sample ( Plasmodium Only) and is very close to the theoretical GC coverage (Theoretical).

    Article Snippet: Preparation of Next-generation Sequencing Libraries DNA extracted from saliva and leukocytes were used to generate SOLiD 4 libraries using NEBNext® Fast DNA Library Prep Set for Ion Torrent (NEB #E6270) with SOLiD 4 adaptors and primers; mixtures of DNA extracted form E. coli K-12 MG1655 and IMR-90 cells were used to generate Ion Torrent libraries using NEBNext Fast DNA Library Prep Set for Ion Torrent (NEB #E6270); DNA extracted from black molly was used to generate Illumina paired end sequencing libraries using NEBNext Ultra DNA Library Prep Kit and Multiplex Oligos for Illumina (NEB #E7370 and #E7335).

    Techniques: Sequencing

    RCA analysis suggests episomal persistence of MCVSyn genomes. Inverted image of a ethidium bromide-stained agarose gel with input material (lanes 1,3,5,7 and 9) or RCA products (lanes 2,4,6,8, 10 and 11) from mock-transfected PFSK-1 cells (lanes 1 and 2), MCVSyn-transfected PFSK-1 cells at 4 days (lanes 3 and 4) or 136 days (lanes 9 and 10) post transfection, the MCC-derived cell lines WaGa or MKL-1 (lanes 5 and 6 or 7 and 8, respectively) or a water control (lane 11). DNA was subjected to restriction enzyme digestion to produce linear, unit-length viral genomes from concatameric RCA products. For size comparison, lane 12 shows unit length MCVSyn genomes excised from plasmid pMK-MCVSyn. The asterisk marks the position of the bacterial pMK plasmid backbone.

    Journal: PLoS Pathogens

    Article Title: A Comprehensive Analysis of Replicating Merkel Cell Polyomavirus Genomes Delineates the Viral Transcription Program and Suggests a Role for mcv-miR-M1 in Episomal Persistence

    doi: 10.1371/journal.ppat.1004974

    Figure Lengend Snippet: RCA analysis suggests episomal persistence of MCVSyn genomes. Inverted image of a ethidium bromide-stained agarose gel with input material (lanes 1,3,5,7 and 9) or RCA products (lanes 2,4,6,8, 10 and 11) from mock-transfected PFSK-1 cells (lanes 1 and 2), MCVSyn-transfected PFSK-1 cells at 4 days (lanes 3 and 4) or 136 days (lanes 9 and 10) post transfection, the MCC-derived cell lines WaGa or MKL-1 (lanes 5 and 6 or 7 and 8, respectively) or a water control (lane 11). DNA was subjected to restriction enzyme digestion to produce linear, unit-length viral genomes from concatameric RCA products. For size comparison, lane 12 shows unit length MCVSyn genomes excised from plasmid pMK-MCVSyn. The asterisk marks the position of the bacterial pMK plasmid backbone.

    Article Snippet: Libraries of RCA products for HTS analysis were generated with the NEBNext Ultra DNA Library Prep Kit for Illumina and sequenced on an Illumina HiSeq2500 instrument.

    Techniques: Staining, Agarose Gel Electrophoresis, Transfection, Derivative Assay, Plasmid Preparation

    Discovery of GRCs in bird species. ( A ) SC spreads of four oscine species immunolabeled with antibodies against SYCP3, the main protein of the lateral element of SC (red), centromere proteins (blue) and MLH1, mismatch repair protein marking recombination sites (green). Arrowheads point to the largest chromosomes ordered according to their size ranks, ZZ (identified by its size and arm ratio), ZW (identified by heteromorphic SC and misaligned centromeres), and GRCs. Arrows in the Insets point to MLH1 foci in GRCs. Micro-GRC bivalents in female barn swallow and European pied flycatcher are indistinguishable from the microchromosomes of the somatic chromosome set. ( B ) DAPI-stained bone marrow cells. ( C ) Reverse and cross-species FISH of GRC DNA probes (green) derived from Bengalese finch (LST), zebra finch (TGU), Eurasian siskin (SSP), and pale martin (RDI) with SC spreads, immunolabeled with antibodies against SYCP3 (red). Centromeres are labeled with antibodies against centromere proteins (blue). Arrowheads point to GRCs and regions on the somatic chromosome set intensely painted with GRC probes in cross-species FISH. Insets show GRCs. The Bengalese finch GRC-specific DNA probe gives a strong signal on the Bengalese finch GRC and slightly paints some regions of the somatic chromosome set. The zebra finch GRC probe paints the distal area of the Bengalese finch GRC and a region of the short arm of SC3. The Eurasian siskin GRC probe paints a micro-GRC of European goldfinch, a region on the long arm of SC3 and some pericentromeric regions. The pale martin GRC probe gives a dispersed signal on the great tit GRC, the ZW bivalent and on SC5. Scale bar: 5 µm.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: From the Cover: Germline-restricted chromosome (GRC) is widespread among songbirds

    doi: 10.1073/pnas.1817373116

    Figure Lengend Snippet: Discovery of GRCs in bird species. ( A ) SC spreads of four oscine species immunolabeled with antibodies against SYCP3, the main protein of the lateral element of SC (red), centromere proteins (blue) and MLH1, mismatch repair protein marking recombination sites (green). Arrowheads point to the largest chromosomes ordered according to their size ranks, ZZ (identified by its size and arm ratio), ZW (identified by heteromorphic SC and misaligned centromeres), and GRCs. Arrows in the Insets point to MLH1 foci in GRCs. Micro-GRC bivalents in female barn swallow and European pied flycatcher are indistinguishable from the microchromosomes of the somatic chromosome set. ( B ) DAPI-stained bone marrow cells. ( C ) Reverse and cross-species FISH of GRC DNA probes (green) derived from Bengalese finch (LST), zebra finch (TGU), Eurasian siskin (SSP), and pale martin (RDI) with SC spreads, immunolabeled with antibodies against SYCP3 (red). Centromeres are labeled with antibodies against centromere proteins (blue). Arrowheads point to GRCs and regions on the somatic chromosome set intensely painted with GRC probes in cross-species FISH. Insets show GRCs. The Bengalese finch GRC-specific DNA probe gives a strong signal on the Bengalese finch GRC and slightly paints some regions of the somatic chromosome set. The zebra finch GRC probe paints the distal area of the Bengalese finch GRC and a region of the short arm of SC3. The Eurasian siskin GRC probe paints a micro-GRC of European goldfinch, a region on the long arm of SC3 and some pericentromeric regions. The pale martin GRC probe gives a dispersed signal on the great tit GRC, the ZW bivalent and on SC5. Scale bar: 5 µm.

    Article Snippet: DNA library for NGS sequencing was prepared using the microdissected GRC DNA libraries using the NEBNext Ultra DNA Library Prep kit (New England Biolabs).

    Techniques: Immunolabeling, Staining, Fluorescence In Situ Hybridization, Derivative Assay, Labeling

    Ala57 allows the mobility of the H3mm7 nucleosome. a T32I and S57A are only the differences between H3.3 and H3mm7. N-terminals of the amino acid sequence of H3.3 and H3mm7 are shown in the illustration of the secondary structure. b High-resolution crystal structure of an H3mm7-containing nucleosome shows the loss of interaction between H3.3S57 and H4R40 in the H3mm7 nucleosome. An overview of the crystal structure of the H3mm7-containing nucleosome (left) and the comparative view around S57 in H3.3 and A57 in H3mm7 (right). c H3mm7 nucleosomes were dissociated at a concentration of 600 mM NaCl. The salt-titration assay of H3mm7, H3.3, and the mutant (H3.3S57A and T32I) containing nucleosomes is shown. The positions of the bands that correspond with the intact nucleosome core particle and naked DNA are indicated on the right. d H3.3S57A and H3mm7 dissociated from the H2A:H2B dimer faster than the other H3 variant-containing nucleosomes. Thermal stability assay of H3mm7, H3.3, and the point mutants H3.3T32I and H3.3S57A. The lines show the fluorescent intensity that indicates nucleosome dissociation at each temperature. The error bars are ± 1 SD ( n = 4). e H3mm7 and H3.3S57A showed a fast histone turnover. FRAP analysis of nucleosomes containing GFP-fused H3mm7, H3.3, and the point mutants. Representative confocal images of FRAP analysis in each H3 variant nucleosome is shown on the left. Plot of average GFP fluorescent recovery rate at each time point after photobleaching is shown on the right. The error bars are ± 1 SD. The number of replicates is indicated in parentheses

    Journal: Nature Communications

    Article Title: Histone H3.3 sub-variant H3mm7 is required for normal skeletal muscle regeneration

    doi: 10.1038/s41467-018-03845-1

    Figure Lengend Snippet: Ala57 allows the mobility of the H3mm7 nucleosome. a T32I and S57A are only the differences between H3.3 and H3mm7. N-terminals of the amino acid sequence of H3.3 and H3mm7 are shown in the illustration of the secondary structure. b High-resolution crystal structure of an H3mm7-containing nucleosome shows the loss of interaction between H3.3S57 and H4R40 in the H3mm7 nucleosome. An overview of the crystal structure of the H3mm7-containing nucleosome (left) and the comparative view around S57 in H3.3 and A57 in H3mm7 (right). c H3mm7 nucleosomes were dissociated at a concentration of 600 mM NaCl. The salt-titration assay of H3mm7, H3.3, and the mutant (H3.3S57A and T32I) containing nucleosomes is shown. The positions of the bands that correspond with the intact nucleosome core particle and naked DNA are indicated on the right. d H3.3S57A and H3mm7 dissociated from the H2A:H2B dimer faster than the other H3 variant-containing nucleosomes. Thermal stability assay of H3mm7, H3.3, and the point mutants H3.3T32I and H3.3S57A. The lines show the fluorescent intensity that indicates nucleosome dissociation at each temperature. The error bars are ± 1 SD ( n = 4). e H3mm7 and H3.3S57A showed a fast histone turnover. FRAP analysis of nucleosomes containing GFP-fused H3mm7, H3.3, and the point mutants. Representative confocal images of FRAP analysis in each H3 variant nucleosome is shown on the left. Plot of average GFP fluorescent recovery rate at each time point after photobleaching is shown on the right. The error bars are ± 1 SD. The number of replicates is indicated in parentheses

    Article Snippet: ChIP-seq ChIP libraries of GFP, H3K27me3, and H3K4me3 were prepared using the NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs) with biological duplicates.

    Techniques: Sequencing, Concentration Assay, Titration, Mutagenesis, Variant Assay, Stability Assay