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Contents of biopolymers of the extracellular polymeric substances from <t>biofilm</t> samples harvested at T5. The <t>DNA</t> content was
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1) Product Images from "Metagenome Survey of a Multispecies and Alga-Associated Biofilm Revealed Key Elements of Bacterial-Algal Interactions in Photobioreactors"

Article Title: Metagenome Survey of a Multispecies and Alga-Associated Biofilm Revealed Key Elements of Bacterial-Algal Interactions in Photobioreactors

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.01641-13

Contents of biopolymers of the extracellular polymeric substances from biofilm samples harvested at T5. The DNA content was
Figure Legend Snippet: Contents of biopolymers of the extracellular polymeric substances from biofilm samples harvested at T5. The DNA content was

Techniques Used:

2) Product Images from "Testing the potential of a ribosomal 16S marker for DNA metabarcoding of insects"

Article Title: Testing the potential of a ribosomal 16S marker for DNA metabarcoding of insects

Journal: PeerJ

doi: 10.7717/peerj.1966

Comparison of 16S Ins (A) and COI Folmer (B) primer performance, both tested with the same 10 bulk samples each containing 52 morphologically distinct macroinvertebrate taxa. Comparison of 16S Ins (A) and COI Folmer (B) primer performance, both tested with the same 10 bulk samples each containing 52 morphologically distinct macroinvertebrate taxa. The 52 taxa are shown on the x -axis with the number of reads obtained with 16S and COI for each sample indicated by black dots on the logarithmic y -axis (mean relative abundance of detected morphotaxa is indicated by red circles). Sequence abundance was normalized across the ten replicates and the amount of tissue used in each DNA extraction. Only OTUs which had minimum abundance of 0.003% in at least one of the 10 samples were included in the analysis. Number of samples for which a morphotaxon was not detected is indicated by orange and red numbers in each plot. A thick vertical line in light red indicates if a morphotaxon was not detected. Detection rates between 16S and COI marker are summarized in a Venn diagram. The availability of 16S reference data from NCBI and own Sanger sequences is indicated by yellow and green background colour behind the taxon names on the x -axis.
Figure Legend Snippet: Comparison of 16S Ins (A) and COI Folmer (B) primer performance, both tested with the same 10 bulk samples each containing 52 morphologically distinct macroinvertebrate taxa. Comparison of 16S Ins (A) and COI Folmer (B) primer performance, both tested with the same 10 bulk samples each containing 52 morphologically distinct macroinvertebrate taxa. The 52 taxa are shown on the x -axis with the number of reads obtained with 16S and COI for each sample indicated by black dots on the logarithmic y -axis (mean relative abundance of detected morphotaxa is indicated by red circles). Sequence abundance was normalized across the ten replicates and the amount of tissue used in each DNA extraction. Only OTUs which had minimum abundance of 0.003% in at least one of the 10 samples were included in the analysis. Number of samples for which a morphotaxon was not detected is indicated by orange and red numbers in each plot. A thick vertical line in light red indicates if a morphotaxon was not detected. Detection rates between 16S and COI marker are summarized in a Venn diagram. The availability of 16S reference data from NCBI and own Sanger sequences is indicated by yellow and green background colour behind the taxon names on the x -axis.

Techniques Used: Sequencing, DNA Extraction, Marker

3) Product Images from "A systems biology approach uncovers a gene co-expression network associated with cell wall degradability in maize"

Article Title: A systems biology approach uncovers a gene co-expression network associated with cell wall degradability in maize

Journal: PLoS ONE

doi: 10.1371/journal.pone.0227011

Selection and characterization of NILs with an introgressed genomic region at QTL positions for cell wall degradability. (A) Experimental workflow used for introgression of F288 alleles at an 18–20 Mb region encompassing the QTL6.05 (referred to as the introgressed QTL6.05 region or QTL6.05 i ) into the background of F271. SNP: single nucleotide polymorphism. (B) The selected BC2S2 NILs 1 (referred to as 1 F271 and 1 F288 ) and NILs 2 (referred to as 2 F271 and 2 F288 ) were grown in the field under well-watered conditions for DNA and mRNA sampling and phenotyping. (C) Comparison of the cell wall degradability IVNDFD performance of NILs introgressed with F288 alleles at QTL6.05 i (blue) and their respective recipient lines with F271 alleles (red). Open circles represent the means of two biological replicates.
Figure Legend Snippet: Selection and characterization of NILs with an introgressed genomic region at QTL positions for cell wall degradability. (A) Experimental workflow used for introgression of F288 alleles at an 18–20 Mb region encompassing the QTL6.05 (referred to as the introgressed QTL6.05 region or QTL6.05 i ) into the background of F271. SNP: single nucleotide polymorphism. (B) The selected BC2S2 NILs 1 (referred to as 1 F271 and 1 F288 ) and NILs 2 (referred to as 2 F271 and 2 F288 ) were grown in the field under well-watered conditions for DNA and mRNA sampling and phenotyping. (C) Comparison of the cell wall degradability IVNDFD performance of NILs introgressed with F288 alleles at QTL6.05 i (blue) and their respective recipient lines with F271 alleles (red). Open circles represent the means of two biological replicates.

Techniques Used: Selection, Sampling

Structure of the introgressed genomic QTL6.05 i region. (A) Large structural variation between B73, F271 and F288 in the introgressed genomic QTL6.05 i region. Dark and light purple boxes represent clusters of QTL in bins 6.05 and 6.07, respectively, as previously defined [ 22 ]. The grey dotted line insertion represents a zoom of the genomic region that included the 1.5 Mb-genomic sequences from F271 and F288. Orange and green lines indicate sense and antisense DNA strands, respectively, of at least 910 bp and sharing at least 98% of identity; arrowed boxes represent genes and pseudogenes. (B) Number of annotated genes supported by RNA-seq predicted genes in the targeted QTL6.05 locus. (C) Gene content distribution in B73, F288 and F271 in the targeted QTL6.05 locus.
Figure Legend Snippet: Structure of the introgressed genomic QTL6.05 i region. (A) Large structural variation between B73, F271 and F288 in the introgressed genomic QTL6.05 i region. Dark and light purple boxes represent clusters of QTL in bins 6.05 and 6.07, respectively, as previously defined [ 22 ]. The grey dotted line insertion represents a zoom of the genomic region that included the 1.5 Mb-genomic sequences from F271 and F288. Orange and green lines indicate sense and antisense DNA strands, respectively, of at least 910 bp and sharing at least 98% of identity; arrowed boxes represent genes and pseudogenes. (B) Number of annotated genes supported by RNA-seq predicted genes in the targeted QTL6.05 locus. (C) Gene content distribution in B73, F288 and F271 in the targeted QTL6.05 locus.

Techniques Used: Genomic Sequencing, RNA Sequencing Assay

4) Product Images from "A Complete Mitochondrial Genome Sequence from a Mesolithic Wild Aurochs (Bos primigenius)"

Article Title: A Complete Mitochondrial Genome Sequence from a Mesolithic Wild Aurochs (Bos primigenius)

Journal: PLoS ONE

doi: 10.1371/journal.pone.0009255

The identity and distribution of DNA nucleotide mismatches in individual Illumina GA reads compared to the CPC98 consensus mtDNA genome. (A) The number and proportion of each nucleotide called in the Illumina GA reads (vertical column) compared to the consensus mtDNA sequence (horizontal column) is presented. (B) Mean percentage of discordant nucleotides for each position across all individual Illumina GA sequence reads.
Figure Legend Snippet: The identity and distribution of DNA nucleotide mismatches in individual Illumina GA reads compared to the CPC98 consensus mtDNA genome. (A) The number and proportion of each nucleotide called in the Illumina GA reads (vertical column) compared to the consensus mtDNA sequence (horizontal column) is presented. (B) Mean percentage of discordant nucleotides for each position across all individual Illumina GA sequence reads.

Techniques Used: Sequencing

5) Product Images from "The Enigma of Progressively Partial Endoreplication: New Insights Provided by Flow Cytometry and Next-Generation Sequencing"

Article Title: The Enigma of Progressively Partial Endoreplication: New Insights Provided by Flow Cytometry and Next-Generation Sequencing

Journal: Genome Biology and Evolution

doi: 10.1093/gbe/evw141

Genome proportion of the most abundant DNA sequence types from 20 largest clusters obtained after graph-based analysis of 2C and 2C + P nuclei. The height of columns represents per cent of reads in the genome of L. discolor . Classification of the clusters is marked with colors.
Figure Legend Snippet: Genome proportion of the most abundant DNA sequence types from 20 largest clusters obtained after graph-based analysis of 2C and 2C + P nuclei. The height of columns represents per cent of reads in the genome of L. discolor . Classification of the clusters is marked with colors.

Techniques Used: Sequencing

Cell cycle analysis using FCM. ( A ) Simplified models of different possible cell cycle progressions. (i) Regular cell cycle, where cells double their nuclear DNA content during the transition from G1-phase through S-phase to G2-phase. ii) Cells replicate their whole DNA content but afterwards some DNA sequences are eliminated. Population with 4C DNA content represents meristematic cells. (iii) Two types of cell cycle occur, one representing PPE cells (2C + P), the other meristematic cells (4C). x axis: nuclear DNA content; y axis: the extent of EdU incorporation into newly synthetized DNA.( B–D ) Cell cycle analysis in root cells of orchid L. discolor ( B, C ) and barley Hordeum vulgare ( D ) using FCM. The roots were pulse-labeled with EdU. x axis represents relative DNA content measured as intensity of DAPI fluorescence (linear scale). y axis shows the extent of EdU incorporation as measured by Alexa Fluor 488 fluorescence intensity (log scale). Different populations represent 2C nuclei, 2C+P nuclei, 4C nuclei, and endopolyploid nuclei (2C+3P, 2C+7P). ( B ) Overall view showing different classes of nuclei. ( C ) The same view with different cell cycles highlighted—PPE (blue), mitotic (yellow), successive endoreplication cycles (red). The green color is caused by the overlap of initial phases of PPE and mitotic cycle. ( D ) Example of a typical “horseshoe” pattern found in barley as a representative of species with regular cell cycle and complete DNA replication.
Figure Legend Snippet: Cell cycle analysis using FCM. ( A ) Simplified models of different possible cell cycle progressions. (i) Regular cell cycle, where cells double their nuclear DNA content during the transition from G1-phase through S-phase to G2-phase. ii) Cells replicate their whole DNA content but afterwards some DNA sequences are eliminated. Population with 4C DNA content represents meristematic cells. (iii) Two types of cell cycle occur, one representing PPE cells (2C + P), the other meristematic cells (4C). x axis: nuclear DNA content; y axis: the extent of EdU incorporation into newly synthetized DNA.( B–D ) Cell cycle analysis in root cells of orchid L. discolor ( B, C ) and barley Hordeum vulgare ( D ) using FCM. The roots were pulse-labeled with EdU. x axis represents relative DNA content measured as intensity of DAPI fluorescence (linear scale). y axis shows the extent of EdU incorporation as measured by Alexa Fluor 488 fluorescence intensity (log scale). Different populations represent 2C nuclei, 2C+P nuclei, 4C nuclei, and endopolyploid nuclei (2C+3P, 2C+7P). ( B ) Overall view showing different classes of nuclei. ( C ) The same view with different cell cycles highlighted—PPE (blue), mitotic (yellow), successive endoreplication cycles (red). The green color is caused by the overlap of initial phases of PPE and mitotic cycle. ( D ) Example of a typical “horseshoe” pattern found in barley as a representative of species with regular cell cycle and complete DNA replication.

Techniques Used: Cell Cycle Assay, Labeling, Fluorescence

6) Product Images from "Comprehensive profiling of retroviral integration sites using target enrichment methods from historical koala samples without an assembled reference genome"

Article Title: Comprehensive profiling of retroviral integration sites using target enrichment methods from historical koala samples without an assembled reference genome

Journal: PeerJ

doi: 10.7717/peerj.1847

Bioinformatic pipeline for identification of KoRV integration sites. The pipeline was run separately for each data set obtained by three different techniques. For the key steps, the number of sequences retained is indicated in parentheses for each technique in this order from left to right: PEC, SPEX and hybridization capture. After processing NGS reads, KoRV integration sites were identified in a two-step analysis of KoRV LTR ends, next to the host DNA flanking KoRV. The first round of selection targeted the A region of the LTR end and its output, was used for subsequent identification of the B region. The LTR ends of all sequences were trimmed off, and only sequences longer than four bp were considered. Using a sequence clustering approach, unique vs. shared integration sites were sorted into clusters. The consensus of each non-singleton cluster was computed using a multiple sequence alignment. These consensus sequences and singleton sequences were queried against wallaby genomic scaffolds and koala Illumina Hiseq reads to determine whether they represented KoRV flanking sequences. At the same time extension products into the KoRV genome were identified.
Figure Legend Snippet: Bioinformatic pipeline for identification of KoRV integration sites. The pipeline was run separately for each data set obtained by three different techniques. For the key steps, the number of sequences retained is indicated in parentheses for each technique in this order from left to right: PEC, SPEX and hybridization capture. After processing NGS reads, KoRV integration sites were identified in a two-step analysis of KoRV LTR ends, next to the host DNA flanking KoRV. The first round of selection targeted the A region of the LTR end and its output, was used for subsequent identification of the B region. The LTR ends of all sequences were trimmed off, and only sequences longer than four bp were considered. Using a sequence clustering approach, unique vs. shared integration sites were sorted into clusters. The consensus of each non-singleton cluster was computed using a multiple sequence alignment. These consensus sequences and singleton sequences were queried against wallaby genomic scaffolds and koala Illumina Hiseq reads to determine whether they represented KoRV flanking sequences. At the same time extension products into the KoRV genome were identified.

Techniques Used: Hybridization, Next-Generation Sequencing, Selection, Sequencing

Experimental work flow for the three enrichment techniques. Abbreviations: HC, Hybridization Capture; PEC, Primer Extension Capture; SPEX, Single Primer Extension. A square of 7 mm × 7 mm of koala skin tissue per museum specimen was extracted in a dedicated ancient DNA (aDNA) facility. A workflow for the three techniques is illustrated. Both HC and PEC require Illumina library preparation as a preliminary step. The double stranded libraries are denatured to single stranded DNA molecules and underwent different experimental procedures in HC and PEC. In HC, single stranded DNA libraries are mixed with magnetic beads immobilized with baits. These are incubated by slow rotation at 65 °C for 48 h. After a series of wash steps, the libraries with non-targets sequences are washed off leaving only the libraries with target sequences hybridized with the baits on beads. These target molecules were then dissociated from the baits using a special elution buffer, and were used as templates for PCR amplification. While in PEC, the singled stranded libraries are mixed with biotinylated oligos for 1 min at 55 °C in which only the libraries with target sequences hybridized with the biotinylated oligos. Primer extension reactions of the biotinylated oligos were performed only to these hybridized libraries. Biotinylated oligos were collected by magnetic beads together with the hybridized targeted libraries. The single stranded libraries with target sequences were dissociated with biotinylated oligos and were eluted for subsequent PCR amplification. In contrast for SPEX, DNA extracts are directly denatured to be single stranded and mixed with the same biotinylated oligos used in PEC for 1 min at 55 °C. Similar as in PEC, primer extension reactions of the biotinylated oligos were performed only to the single molecules (target sequences) hybridized with biotinylated oligos. These hybrid molecules were collected using magnetic beads. The original single stranded target molecules were washed away and the biotinylated oligos with 3′ extension were eluted off the beads and were treated with a poly C tailing reaction. These poly C tailed molecules were amplified using primers with a 5′ overhang of the Illumina sequencing adaptor. Through this process, the SPEX products were constructed into Illumina libraries without an additional library preparation step. These SPEX-Illumina libraries were then used in an index PCR and a further amplification step. As shown, SPEX requires at least one more amplification step than HC or PEC, which may explain the high level of clonality in the SPEX result.
Figure Legend Snippet: Experimental work flow for the three enrichment techniques. Abbreviations: HC, Hybridization Capture; PEC, Primer Extension Capture; SPEX, Single Primer Extension. A square of 7 mm × 7 mm of koala skin tissue per museum specimen was extracted in a dedicated ancient DNA (aDNA) facility. A workflow for the three techniques is illustrated. Both HC and PEC require Illumina library preparation as a preliminary step. The double stranded libraries are denatured to single stranded DNA molecules and underwent different experimental procedures in HC and PEC. In HC, single stranded DNA libraries are mixed with magnetic beads immobilized with baits. These are incubated by slow rotation at 65 °C for 48 h. After a series of wash steps, the libraries with non-targets sequences are washed off leaving only the libraries with target sequences hybridized with the baits on beads. These target molecules were then dissociated from the baits using a special elution buffer, and were used as templates for PCR amplification. While in PEC, the singled stranded libraries are mixed with biotinylated oligos for 1 min at 55 °C in which only the libraries with target sequences hybridized with the biotinylated oligos. Primer extension reactions of the biotinylated oligos were performed only to these hybridized libraries. Biotinylated oligos were collected by magnetic beads together with the hybridized targeted libraries. The single stranded libraries with target sequences were dissociated with biotinylated oligos and were eluted for subsequent PCR amplification. In contrast for SPEX, DNA extracts are directly denatured to be single stranded and mixed with the same biotinylated oligos used in PEC for 1 min at 55 °C. Similar as in PEC, primer extension reactions of the biotinylated oligos were performed only to the single molecules (target sequences) hybridized with biotinylated oligos. These hybrid molecules were collected using magnetic beads. The original single stranded target molecules were washed away and the biotinylated oligos with 3′ extension were eluted off the beads and were treated with a poly C tailing reaction. These poly C tailed molecules were amplified using primers with a 5′ overhang of the Illumina sequencing adaptor. Through this process, the SPEX products were constructed into Illumina libraries without an additional library preparation step. These SPEX-Illumina libraries were then used in an index PCR and a further amplification step. As shown, SPEX requires at least one more amplification step than HC or PEC, which may explain the high level of clonality in the SPEX result.

Techniques Used: Flow Cytometry, Hybridization, Ancient DNA Assay, Magnetic Beads, Incubation, Polymerase Chain Reaction, Amplification, Sequencing, Construct

7) Product Images from "Dry and wet approaches for genome-wide functional annotation of conventional and unconventional transcriptional activators"

Article Title: Dry and wet approaches for genome-wide functional annotation of conventional and unconventional transcriptional activators

Journal: Computational and Structural Biotechnology Journal

doi: 10.1016/j.csbj.2016.06.004

Heterologous in vivo approaches for TF DNA binding site identification. a. The yeast one-hybrid (Y1H) is a DNA-centered approach used to identify TFs capable of binding to a specific DNA element. The DNA sequence to be interrogated (“DNA bait”) is cloned into a selectable yeast plasmid, upstream of reporter genes such as HIS3 and LacZ , and subsequently integrated into a mutated marker locus within the yeast genome. A TF of interest (either a selected one or a whole cDNA library; green ) is expressed as a fusion with the yeast Gal4 activation domain (Gal4 AD, shown in blue ). Positive hits (i.e., TFs bearing a DBD capable of interacting with the bait sequence) activate reporter gene expression. The transcriptional machinery is in red . TATA: TATA box. b. The bacterial one-hybrid (B1H) is a TF-centered approach used to identify the DNA element bound by a (putative) TF or activator. A bi-cistronic vector bearing a randomized region ( rainbowed ) upstream of two reporter genes ( HIS3 and URA3 ) is used as a “prey” to identify the DNA elements bound by the “bait” TF (or putative activator) (shown in green ) fused to the ω subunit ( blue ) of bacterial RNA polymerase ( orange ). The yeast URA3 gene is used as negative selection marker (5-FOA counter-selection) to eliminate self-activating DNA elements; the yeast HIS3 gene is used as a positive selection marker to identify the DNA elements bound by the bait TF. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Figure Legend Snippet: Heterologous in vivo approaches for TF DNA binding site identification. a. The yeast one-hybrid (Y1H) is a DNA-centered approach used to identify TFs capable of binding to a specific DNA element. The DNA sequence to be interrogated (“DNA bait”) is cloned into a selectable yeast plasmid, upstream of reporter genes such as HIS3 and LacZ , and subsequently integrated into a mutated marker locus within the yeast genome. A TF of interest (either a selected one or a whole cDNA library; green ) is expressed as a fusion with the yeast Gal4 activation domain (Gal4 AD, shown in blue ). Positive hits (i.e., TFs bearing a DBD capable of interacting with the bait sequence) activate reporter gene expression. The transcriptional machinery is in red . TATA: TATA box. b. The bacterial one-hybrid (B1H) is a TF-centered approach used to identify the DNA element bound by a (putative) TF or activator. A bi-cistronic vector bearing a randomized region ( rainbowed ) upstream of two reporter genes ( HIS3 and URA3 ) is used as a “prey” to identify the DNA elements bound by the “bait” TF (or putative activator) (shown in green ) fused to the ω subunit ( blue ) of bacterial RNA polymerase ( orange ). The yeast URA3 gene is used as negative selection marker (5-FOA counter-selection) to eliminate self-activating DNA elements; the yeast HIS3 gene is used as a positive selection marker to identify the DNA elements bound by the bait TF. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Techniques Used: In Vivo, Binding Assay, Sequencing, Clone Assay, Plasmid Preparation, Marker, cDNA Library Assay, Activation Assay, Expressing, Selection

Identification and functional validation of TFs and unconventional activators. a. Schematic representation of the transcriptional activator trap (TAT) approach, as applied to the identification and functional validation of AD-containing, conventional and unconventional transcriptional activators. Reporter gene expression ( HIS3 , URA3 and LacZ ) is activated if the query TF (a selected subset or a whole cDNA library; green ) fused to the Gal4-DBD ( blue ) behaves as a transcriptional activator — i.e., it is capable of recruiting RNA Pol II transcription machinery ( red ). UAS: upstream activating sequence (Gal4 DNA-binding site); TATA: TATA box. b. Nuclear transportation trap (NTT) assay used to test the autonomous nuclear localization capacity of putative unconventional activators. A chimeric protein (NLS-less TF, blue ) comprising a modified bacterial DBD (LexA), a portion of the E. coli maltose binding protein and the yeast Gal4 AD, but lacking a nuclear localization signal (NLS), is fused to a candidate unconventional activator (UA, green ). If the latter contains a NLS (either recognizable in silico or cryptic), it will direct the chimeric protein to the nucleus, thus leading to reporter gene ( HIS3 , LacZ ) activation. The transcriptional machinery is in red . LBS: LexA binding site; TATA: TATA box. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Figure Legend Snippet: Identification and functional validation of TFs and unconventional activators. a. Schematic representation of the transcriptional activator trap (TAT) approach, as applied to the identification and functional validation of AD-containing, conventional and unconventional transcriptional activators. Reporter gene expression ( HIS3 , URA3 and LacZ ) is activated if the query TF (a selected subset or a whole cDNA library; green ) fused to the Gal4-DBD ( blue ) behaves as a transcriptional activator — i.e., it is capable of recruiting RNA Pol II transcription machinery ( red ). UAS: upstream activating sequence (Gal4 DNA-binding site); TATA: TATA box. b. Nuclear transportation trap (NTT) assay used to test the autonomous nuclear localization capacity of putative unconventional activators. A chimeric protein (NLS-less TF, blue ) comprising a modified bacterial DBD (LexA), a portion of the E. coli maltose binding protein and the yeast Gal4 AD, but lacking a nuclear localization signal (NLS), is fused to a candidate unconventional activator (UA, green ). If the latter contains a NLS (either recognizable in silico or cryptic), it will direct the chimeric protein to the nucleus, thus leading to reporter gene ( HIS3 , LacZ ) activation. The transcriptional machinery is in red . LBS: LexA binding site; TATA: TATA box. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Techniques Used: Functional Assay, Expressing, cDNA Library Assay, Sequencing, Binding Assay, Modification, In Silico, Activation Assay

8) Product Images from "Specificity landscapes unmask submaximal binding site preferences of transcription factors"

Article Title: Specificity landscapes unmask submaximal binding site preferences of transcription factors

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

doi: 10.1073/pnas.1811431115

SEL/DiSEL to compare different high-throughput experimental platforms. SELs ( Left ), scatterplots of quantile-normalized DNA binding intensities for all 8-mers ( Center ), and DiSELs ( Right ) comparing DPIs obtained through high-throughput platforms with PBM. ( A ) CSI vs. PBM data for Saccharomyces cerevisiae Gzf3 (seed motif: 5′GATAAG3′). ( B ) HiTS-FLIP vs. PBM data for S. cerevisiae Gcn4 (seed motif: 5′TGACTCA3′). ( C ) MITOMI vs. PBM data for S. cerevisiae Cbf1 (seed motif: 5′CACGTG3′). ( D ) HT-SELEX for Homo sapiens FOXJ3 vs. PBM for Mus musculus Foxj3 (seed motif: 5′AAACA3′). DNA logos derived from PBM were downloaded from UniPROBE. SEL peaks represent DNA binding preferences of the protein as measured by the experimental platform, whereas DiSEL peaks correspond to binding preference identified by one platform but not the other. Few differences are pointed out on DiSELs (arrows). All data are displayed as z scores.
Figure Legend Snippet: SEL/DiSEL to compare different high-throughput experimental platforms. SELs ( Left ), scatterplots of quantile-normalized DNA binding intensities for all 8-mers ( Center ), and DiSELs ( Right ) comparing DPIs obtained through high-throughput platforms with PBM. ( A ) CSI vs. PBM data for Saccharomyces cerevisiae Gzf3 (seed motif: 5′GATAAG3′). ( B ) HiTS-FLIP vs. PBM data for S. cerevisiae Gcn4 (seed motif: 5′TGACTCA3′). ( C ) MITOMI vs. PBM data for S. cerevisiae Cbf1 (seed motif: 5′CACGTG3′). ( D ) HT-SELEX for Homo sapiens FOXJ3 vs. PBM for Mus musculus Foxj3 (seed motif: 5′AAACA3′). DNA logos derived from PBM were downloaded from UniPROBE. SEL peaks represent DNA binding preferences of the protein as measured by the experimental platform, whereas DiSEL peaks correspond to binding preference identified by one platform but not the other. Few differences are pointed out on DiSELs (arrows). All data are displayed as z scores.

Techniques Used: High Throughput Screening Assay, Binding Assay, Derivative Assay

9) Product Images from "Human centromeric CENP-A chromatin is a homotypic, octameric nucleosome at all cell cycle points"

Article Title: Human centromeric CENP-A chromatin is a homotypic, octameric nucleosome at all cell cycle points

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201608083

High-throughput sequencing and mapping to α-satellite DNA reveals that centromeric CENP-A chromatin is an octameric nucleosome with transient DNA unwrapping of the DNA entry and exit sites at G1, G2, and mitosis. (A) Experimental design for obtaining CENP-A TAP – and H3.1 TAP -bound DNA sequences. (B) Microcapillary electrophoresis of MNase-digested bulk input mononucleosomes (top) and CENP-A TAP or H3.1 TAP or NH2 H3 CATD native ChIP (middle and bottom, immunoprecipitation). Purified CENP-A chromatin is nucleosome-like but protects a shorter DNA length than does H3.1-containing nucleosomes. (C) Quantitative real-time PCR for α-satellite DNA extracted from CENP-A TAP chromatin in random cycling (RC; magenta), G1 (red), and G2 (blue). n = 2 from two independent replicates. Error bars represent SEM. (D) CENP-A TAP – and H3.1 TAP -bound DNA was sequenced using paired-end 100-bp ChIP sequencing and then mapped to the human genome 38 assembly (hg38), which contains α-satellite sequence models for each centromere. Approximately 50% of CENP-A TAP –bound DNA is centromeric at G1 and G2. (E) CENP-A TAP –bound DNA sequences that mapped to α-satellite DNA were analyzed for their nucleosomal DNA length and overlaid on the microcapillary electrophoresis data (black line) shown in B. (F) CENP-A TAP –bound DNA sequences that mapped to α-satellite DNA on chromosome X were analyzed for their nucleosomal DNA length and overlaid on the microcapillary electrophoresis data (black line) shown in B. (G) Distribution of the chromatin DNA lengths into two length bins: subnucleosomal (60–85 bp, as predicted for tetrasomes and hemisomes; Hasson et al., 2013 ) and nucleosomal (100–170 bp) for bulk input chromatin and affinity-purified CENP-A TAP and H3.1 TAP chromatin mapped to α-satellite DNA. n = 2 from two independent replicates. Error bars represent SEM.
Figure Legend Snippet: High-throughput sequencing and mapping to α-satellite DNA reveals that centromeric CENP-A chromatin is an octameric nucleosome with transient DNA unwrapping of the DNA entry and exit sites at G1, G2, and mitosis. (A) Experimental design for obtaining CENP-A TAP – and H3.1 TAP -bound DNA sequences. (B) Microcapillary electrophoresis of MNase-digested bulk input mononucleosomes (top) and CENP-A TAP or H3.1 TAP or NH2 H3 CATD native ChIP (middle and bottom, immunoprecipitation). Purified CENP-A chromatin is nucleosome-like but protects a shorter DNA length than does H3.1-containing nucleosomes. (C) Quantitative real-time PCR for α-satellite DNA extracted from CENP-A TAP chromatin in random cycling (RC; magenta), G1 (red), and G2 (blue). n = 2 from two independent replicates. Error bars represent SEM. (D) CENP-A TAP – and H3.1 TAP -bound DNA was sequenced using paired-end 100-bp ChIP sequencing and then mapped to the human genome 38 assembly (hg38), which contains α-satellite sequence models for each centromere. Approximately 50% of CENP-A TAP –bound DNA is centromeric at G1 and G2. (E) CENP-A TAP –bound DNA sequences that mapped to α-satellite DNA were analyzed for their nucleosomal DNA length and overlaid on the microcapillary electrophoresis data (black line) shown in B. (F) CENP-A TAP –bound DNA sequences that mapped to α-satellite DNA on chromosome X were analyzed for their nucleosomal DNA length and overlaid on the microcapillary electrophoresis data (black line) shown in B. (G) Distribution of the chromatin DNA lengths into two length bins: subnucleosomal (60–85 bp, as predicted for tetrasomes and hemisomes; Hasson et al., 2013 ) and nucleosomal (100–170 bp) for bulk input chromatin and affinity-purified CENP-A TAP and H3.1 TAP chromatin mapped to α-satellite DNA. n = 2 from two independent replicates. Error bars represent SEM.

Techniques Used: Next-Generation Sequencing, Electrophoresis, Chromatin Immunoprecipitation, Immunoprecipitation, Purification, Real-time Polymerase Chain Reaction, Sequencing, Affinity Purification

CENP-A chromatin has the physical characteristics of nucleosomes. (A) Experimental design for the separation of in vitro–reconstituted octameric nucleosomes or tetrasomes over a 5–25% sucrose gradient. (B) After sedimentation of in vitro–reconstituted H3 or CENP-A octameric nucleosomes or (CENP-A/H4) 2 or (H3/H4) 2 tetrasomes, fractions were immunoblotted for CENP-A or H3. (C) Experimental design for the sedimentation of in vivo bulk nucleosomes and short polynucleosomes at different points of the cell cycle. (D) Moderate MNase digestion profile of bulk chromatin from random cycling (RC) cells expressing CENP-A TAP or H3.1 TAP . (E) Sucrose gradient sedimentation and fractionation of bulk nucleosomes from CENP-A TAP –expressing cells synchronized at G1, G2, and mitosis. (top) Ethidium bromide stained DNA agarose gel revealing the DNA length extracted from the different fractions. (bottom) Immunoblot for CENP-A TAP . (F) Real-time quantitative PCR for α-satellite DNA in the different fractions (colored). Second axis shows quantification of CENP-A immunoblot shown in E. No CENP-A or α-satellite DNA was detected in fractions 7 and 9.
Figure Legend Snippet: CENP-A chromatin has the physical characteristics of nucleosomes. (A) Experimental design for the separation of in vitro–reconstituted octameric nucleosomes or tetrasomes over a 5–25% sucrose gradient. (B) After sedimentation of in vitro–reconstituted H3 or CENP-A octameric nucleosomes or (CENP-A/H4) 2 or (H3/H4) 2 tetrasomes, fractions were immunoblotted for CENP-A or H3. (C) Experimental design for the sedimentation of in vivo bulk nucleosomes and short polynucleosomes at different points of the cell cycle. (D) Moderate MNase digestion profile of bulk chromatin from random cycling (RC) cells expressing CENP-A TAP or H3.1 TAP . (E) Sucrose gradient sedimentation and fractionation of bulk nucleosomes from CENP-A TAP –expressing cells synchronized at G1, G2, and mitosis. (top) Ethidium bromide stained DNA agarose gel revealing the DNA length extracted from the different fractions. (bottom) Immunoblot for CENP-A TAP . (F) Real-time quantitative PCR for α-satellite DNA in the different fractions (colored). Second axis shows quantification of CENP-A immunoblot shown in E. No CENP-A or α-satellite DNA was detected in fractions 7 and 9.

Techniques Used: In Vitro, Sedimentation, In Vivo, Expressing, Fractionation, Staining, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction

CENP-A chromatin is nucleosome-like but with transient DNA unwrapping of the DNA entry and exit sites before (G1) and after (G2) DNA replication at all human centromeres. CENP-A TAP –bound DNA sequences that mapped, using human centromere reference models for the centromeres for the 22 human autosomes (unpublished data), to α-satellite DNA throughout each centromere were analyzed for DNA lengths protected from micrococcal nuclease digestion.
Figure Legend Snippet: CENP-A chromatin is nucleosome-like but with transient DNA unwrapping of the DNA entry and exit sites before (G1) and after (G2) DNA replication at all human centromeres. CENP-A TAP –bound DNA sequences that mapped, using human centromere reference models for the centromeres for the 22 human autosomes (unpublished data), to α-satellite DNA throughout each centromere were analyzed for DNA lengths protected from micrococcal nuclease digestion.

Techniques Used:

10) Product Images from "Using defined finger-finger interfaces as units of assembly for constructing zinc-finger nucleases"

Article Title: Using defined finger-finger interfaces as units of assembly for constructing zinc-finger nucleases

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks1357

Selection of GANNAG finger sets. ( A ) Schematic of two-finger ZFP library used in these selections with the specificity determinants mapped to their recognition positions in their binding site. The dashed box indicates the position of the dinucleotide junction. This library contains randomized amino acids at the finger–finger interface at positions +5 and +6 of finger 1 (randomized with VNS codons) and positions −1, +1 and +2 of finger 2 (randomized with NNW codons), where the numbering scheme refers to the position of the residue relative to the start of the recognition helix. The finger 1 residues at positions −1, 1 and 2 (R, S and D) represent the N-terminal cap, and the finger 2 residues at positions 5 and 6 (T and R) represent the C-terminal cap. ( B ) Schematic representation of the two-stage process used to identify two-finger modules with the desired sequence preference. In Stage 1, the B1H system is used to select two-finger modules complementary to each target site. The randomized two-finger module library is fused between the DNA-binding domain of the Engrailed homeodomain and the ω-subunit of the RNA polymerase. The fixed 6-bp GANNAG target site is present on the His3/Ura3 reporter plasmid between the homeodomain binding site and the −35 box. In Stage 2, the DNA binding specificity of candidate two-finger modules obtained from the first stage of the selection are interrogated. Each two-finger module is fused to an N-terminal finger (RSDTLAR) that binds to the ‘GCG’ triplet adjacent to the 6 bp randomized zinc-finger binding region on the reporter plasmid. The recovered binding sites are determined by Illumina sequencing, and then a binding site motif is calculated from these sequences ( 56 ).
Figure Legend Snippet: Selection of GANNAG finger sets. ( A ) Schematic of two-finger ZFP library used in these selections with the specificity determinants mapped to their recognition positions in their binding site. The dashed box indicates the position of the dinucleotide junction. This library contains randomized amino acids at the finger–finger interface at positions +5 and +6 of finger 1 (randomized with VNS codons) and positions −1, +1 and +2 of finger 2 (randomized with NNW codons), where the numbering scheme refers to the position of the residue relative to the start of the recognition helix. The finger 1 residues at positions −1, 1 and 2 (R, S and D) represent the N-terminal cap, and the finger 2 residues at positions 5 and 6 (T and R) represent the C-terminal cap. ( B ) Schematic representation of the two-stage process used to identify two-finger modules with the desired sequence preference. In Stage 1, the B1H system is used to select two-finger modules complementary to each target site. The randomized two-finger module library is fused between the DNA-binding domain of the Engrailed homeodomain and the ω-subunit of the RNA polymerase. The fixed 6-bp GANNAG target site is present on the His3/Ura3 reporter plasmid between the homeodomain binding site and the −35 box. In Stage 2, the DNA binding specificity of candidate two-finger modules obtained from the first stage of the selection are interrogated. Each two-finger module is fused to an N-terminal finger (RSDTLAR) that binds to the ‘GCG’ triplet adjacent to the 6 bp randomized zinc-finger binding region on the reporter plasmid. The recovered binding sites are determined by Illumina sequencing, and then a binding site motif is calculated from these sequences ( 56 ).

Techniques Used: Selection, Binding Assay, Sequencing, Plasmid Preparation

Related Articles

Amplification:

Article Title: Epigenetic profiling for the molecular classification of metastatic brain tumors
Article Snippet: .. All the samples passing the quality control test were whole-genome amplified, enzymatically fragmented and repaired using the Infinium HD FFPE DNA Restore kit (WG-321-1002, Illumina Inc., San Diego, CA, USA). .. Finally, the fragmented and restored sodium bisulfite-modified DNA specimens were hybridized into the HM450K BeadChips and scanned using the Illumina iScan microarray scanner following the manufacturer’s recommendations (Illumina Inc., San Diego, CA, USA).

Article Title: IDH2 Mutations Define a Unique Subtype of Breast Cancer with Altered Nuclear Polarity
Article Snippet: .. DNA samples from five IDH2 -mutant SPCRPs (cases 3-5, 7, and 9), one TET2 -mutant SPCRP (case 11) and two invasive ductal carcinomas of no special type (IDCs, one ER-positive/HER2-negative and one ER-negative/HER2-negative) were bisulfite-converted using the EZ-96 DNA Methylation Kit (Zymo Research), restored using the Illumina Infinium HD FFPE DNA Restore Kit, and whole-genome amplified ( ). .. The BeadChips were scanned, and the raw data files containing the fluorescence intensity data for each probe were generated.

Article Title: Patient-Specific Screening Using High-Grade Glioma Explants to Determine Potential Radiosensitization by a TGF-β Small Molecule Inhibitor
Article Snippet: .. After the unmethylated cytosines were deaminated, the DNA then underwent restoration using the Illumina Infinium HD FFPE DNA Restore Kit, followed by whole-genome overnight amplification. .. The amplified DNA was then enzymatically fragmented using end-point fragmentation, isolated, precipitated in isopropanol, and resuspended in Illumina RA1 buffer.

Formalin-fixed Paraffin-Embedded:

Article Title: Epigenetic profiling for the molecular classification of metastatic brain tumors
Article Snippet: .. All the samples passing the quality control test were whole-genome amplified, enzymatically fragmented and repaired using the Infinium HD FFPE DNA Restore kit (WG-321-1002, Illumina Inc., San Diego, CA, USA). .. Finally, the fragmented and restored sodium bisulfite-modified DNA specimens were hybridized into the HM450K BeadChips and scanned using the Illumina iScan microarray scanner following the manufacturer’s recommendations (Illumina Inc., San Diego, CA, USA).

Article Title: Expanding epigenomics to archived FFPE tissues: An evaluation of DNA repair methodologies
Article Snippet: .. Infinium HD FFPE DNA Restore kit (Illumina, Inc., San Diego, CA) ( ) protocol was carried out according to the manufacturer’s instructions on 8µl of bisulfite-treated FFPE DNA (RES1, RES2, & RES3). .. The DNA was eluted with DiH2 0 after a 5 minute incubation and stored at −20°C prior to the Infinium processing.

Article Title: IDH2 Mutations Define a Unique Subtype of Breast Cancer with Altered Nuclear Polarity
Article Snippet: .. DNA samples from five IDH2 -mutant SPCRPs (cases 3-5, 7, and 9), one TET2 -mutant SPCRP (case 11) and two invasive ductal carcinomas of no special type (IDCs, one ER-positive/HER2-negative and one ER-negative/HER2-negative) were bisulfite-converted using the EZ-96 DNA Methylation Kit (Zymo Research), restored using the Illumina Infinium HD FFPE DNA Restore Kit, and whole-genome amplified ( ). .. The BeadChips were scanned, and the raw data files containing the fluorescence intensity data for each probe were generated.

Article Title: Unraveling transformation of follicular lymphoma to diffuse large B-cell lymphoma
Article Snippet: .. SNP arrays The DNAs were first subjected to the Infinium HD FFPE DNA Restore Protocol (Illumina) and up to 200 ng DNA per sample were hybridized for the SNP analysis. .. Genotyping was conducted using the Illumina HumanOmni2.5 BeadChip according to the manufacturer's protocols (Illumina).

Article Title: Validation of the MethylationEPIC BeadChip for fresh-frozen and formalin-fixed paraffin-embedded tumours
Article Snippet: .. DNA restoration—Infinium All of eluate 1 of the bisulfite-converted unrestored FFPE DNA (n = 4 samples) was used for restoration with the Infinium HD FFPE DNA Restore Kit (WG-321-1002, Illumina) using the Infinium HD FFPE Restore Protocol supplied by the manufacturer. .. Genome-wide methylation arrays Infinium HumanMethylation450 BeadChips and Infinium MethylationEPIC BeadChips (Illumina) were used for the determination of methylation levels of more than 450,000 and 850,000 CpG sites, respectively, as previously described [ ].

Article Title: Patient-Specific Screening Using High-Grade Glioma Explants to Determine Potential Radiosensitization by a TGF-β Small Molecule Inhibitor
Article Snippet: .. After the unmethylated cytosines were deaminated, the DNA then underwent restoration using the Illumina Infinium HD FFPE DNA Restore Kit, followed by whole-genome overnight amplification. .. The amplified DNA was then enzymatically fragmented using end-point fragmentation, isolated, precipitated in isopropanol, and resuspended in Illumina RA1 buffer.

Article Title: Bead-linked transposomes enable a normalization-free workflow for NGS library preparation
Article Snippet: .. Due to the degraded nature of the sample, we made three adjustments to the workflow: first, the DNA was ‘repaired’ with a standard FFPE repair kit (Illumina, cat. no. WG-321-1002) to improve DNA quality; second, we increased the number of PCR cycles from 5 to 8 to improve yield; third, because we expect smaller fragments, we used a single-sided SPRI size selection with 1.8× volume of SPBs to increase yield and purify the smaller fragments expected from tagmentation of degraded DNA. ..

Mutagenesis:

Article Title: IDH2 Mutations Define a Unique Subtype of Breast Cancer with Altered Nuclear Polarity
Article Snippet: .. DNA samples from five IDH2 -mutant SPCRPs (cases 3-5, 7, and 9), one TET2 -mutant SPCRP (case 11) and two invasive ductal carcinomas of no special type (IDCs, one ER-positive/HER2-negative and one ER-negative/HER2-negative) were bisulfite-converted using the EZ-96 DNA Methylation Kit (Zymo Research), restored using the Illumina Infinium HD FFPE DNA Restore Kit, and whole-genome amplified ( ). .. The BeadChips were scanned, and the raw data files containing the fluorescence intensity data for each probe were generated.

DNA Methylation Assay:

Article Title: IDH2 Mutations Define a Unique Subtype of Breast Cancer with Altered Nuclear Polarity
Article Snippet: .. DNA samples from five IDH2 -mutant SPCRPs (cases 3-5, 7, and 9), one TET2 -mutant SPCRP (case 11) and two invasive ductal carcinomas of no special type (IDCs, one ER-positive/HER2-negative and one ER-negative/HER2-negative) were bisulfite-converted using the EZ-96 DNA Methylation Kit (Zymo Research), restored using the Illumina Infinium HD FFPE DNA Restore Kit, and whole-genome amplified ( ). .. The BeadChips were scanned, and the raw data files containing the fluorescence intensity data for each probe were generated.

Selection:

Article Title: Bead-linked transposomes enable a normalization-free workflow for NGS library preparation
Article Snippet: .. Due to the degraded nature of the sample, we made three adjustments to the workflow: first, the DNA was ‘repaired’ with a standard FFPE repair kit (Illumina, cat. no. WG-321-1002) to improve DNA quality; second, we increased the number of PCR cycles from 5 to 8 to improve yield; third, because we expect smaller fragments, we used a single-sided SPRI size selection with 1.8× volume of SPBs to increase yield and purify the smaller fragments expected from tagmentation of degraded DNA. ..

Polymerase Chain Reaction:

Article Title: Bead-linked transposomes enable a normalization-free workflow for NGS library preparation
Article Snippet: .. Due to the degraded nature of the sample, we made three adjustments to the workflow: first, the DNA was ‘repaired’ with a standard FFPE repair kit (Illumina, cat. no. WG-321-1002) to improve DNA quality; second, we increased the number of PCR cycles from 5 to 8 to improve yield; third, because we expect smaller fragments, we used a single-sided SPRI size selection with 1.8× volume of SPBs to increase yield and purify the smaller fragments expected from tagmentation of degraded DNA. ..

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    Illumina Inc traditional rna sequencing library preparation
    Cross-platform comparison of competitive amplicon library preparation with <t>TaqMan</t> qPCR and Illumina <t>RNA-Sequencing.</t> a ) Comparison of TaqMan qPCR with competitive amplicon library preparation (n = 146) for samples A and B without correction for systematic biases. Data is normalized to a median relative abundance. b ) Comparison of Illumina RNA-Sequencing with competitive amplicon library preparation (n = 170) for samples A and B without correction for systematic biases. Data is normalized to a median relative abundance. For a) and b), Spearman’s rank correlation coefficient is noted (r s ). The average of differences for measurements of samples A and B between competitive amplicon library preparation and TaqMan qPCR ( Figure S1 ) or Illumina RNA-sequencing ( Figure S2 ) was determined for each endogenous target; and to illustrate the systematic bias away from the regression line, data points for MMP2 have been highlighted in orange. This difference was subtracted from TaqMan qPCR or Illumina RNA-sequencing measurements for samples C and D and plotted (X-axis). Competitive amplicon library preparation measurements of C and D are plotted on the Y-axis. c) Comparison of TaqMan qPCR with competitive amplicon library preparation (n = 146) for samples C and D with correction for platform and assay specific bias. d) Comparison of Illumina RNA-Sequencing with competitive amplicon library preparation (n = 170) for samples C and D with correction for platform and assay specific bias.
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    Illumina Inc ss dna sequencing
    ( a ) Schematic representation of the <t>DNA</t> oligonucleotides from which <t>Illumina</t> libraries were generated. ( b ) Distribution of duplex lengths amongst a sequenced sample of single-stranded DNA oligonucleotides. We extracted reads corresponding to the oligonucleotide by searching the output data for the 12 nt known sequence tag. Duplex lengths were then calculated by counting the number of bases at the 3′-end found to be the reverse complement of those at the 5′-end. This revealed a smooth distribution of values over a range of sizes, with large peaks at 12 bp (likely resulting from intermolecular annealing) and 9 bp (probably the consequence of a 3 bp duplex that can form between the known sequence tag and RNA binding site). ( c ) Two proposed mechanisms for the formation of species with 9 nt of reverse complementarity between the 5′ and 3′-ends, the most common duplex length observed. The vast majority of these were found to have 3 bp ‘seed duplexes’ formed by base pairing between –CGT– in the sequence tag and either the –GCA– in the 3′ half of the RNA oligonucleotide binding site, or the CA-dinucleotide at the start of the binding site when the preceding nucleotide (the last of the 19 nt random sequence) was G.
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    Regulation of de novo lipid synthesis in M . alpina . Pathways of de novo lipid synthesis are illustrated. Major intermediates and final products are highlighted in black, and enzymes found to be regulated at the <t>RNA</t> level during lipid synthesis are indicated by circled numbers. Enzymes involved in glycolysis are highlighted in light blue, pentose phosphate pathway in pink, tricarboxylic acid cycle in yellow, fatty acid synthesis in dark blue and glycerolipid synthesis in orange. <t>Transcriptome</t> analysis was performed in samples from 12, 2 and 0.5 h prior to and 1, 12 and 48 h after nitrogen depletion. RNA expression levels (FPKM in log 10 scale) for up-regulated enzymes are plotted in red and down-regulated enzymes in green. PK (EC 2.7.1.40): Pyruvate kinase, PCK (EC 4.1.1.32 and EC 4.1.1.49): Phosphoenolpyruvate carboxykinase, HK (EC 2.7.1.1 and EC 2.7.1.2): Hexokinase, ALDO (EC 4.1.2.13): Fructose 1,6-bisphosphate aldolase, GAPDH (EC 1.2.1.12): Glyceraldehyde 3-phosphate dehydrogenase, ENO (EC 4.2.1.11): 2-phosphoglycerate dehydratase, ACO (EC 4.2.1.3): Aconitase, IDH (EC 1.1.1.42): Isocitrate dehydrogenase, PDH (EC 1.2.4.1): Pyruvate dehydrogenase, OGDH (EC 1.2.4.2): Oxoglutarate dehydrogenase , SUCLG (EC 6.2.1.4): Succinyl-CoA synthetase, MDH (EC 1.1.1.37): Malate dehydrogenase, ACLY (EC 2.3.3.8): ATP-citric lyase, ACS (EC 6.2.1.1): Acetyl-CoA synthetase, ALDH (EC 1.2.1.3): Aldehyde dehydrogenase, ADH (EC 1.1.1.2): Alcohol dehydrogenase, GPAT (EC 2.3.1.15): 3-glycerophosphate acyltransferase, DGAT (EC 2.3.1.20): Diacylglycerol O -acyltransferase, ACSL (EC 6.2.1.3): Acyl-CoA synthetase, CRAT (EC 2.3.1.7): Carnitine O-acetyltransferase, LIP (EC 3.1.1.3): Triacylglycerol lipase, G6PD (EC 1.1.1.49): Glucose-6-phosphate dehydrogenase and PGD (EC 1.1.1.44): Phosphogluconate dehydrogenase.
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    Cross-platform comparison of competitive amplicon library preparation with TaqMan qPCR and Illumina RNA-Sequencing. a ) Comparison of TaqMan qPCR with competitive amplicon library preparation (n = 146) for samples A and B without correction for systematic biases. Data is normalized to a median relative abundance. b ) Comparison of Illumina RNA-Sequencing with competitive amplicon library preparation (n = 170) for samples A and B without correction for systematic biases. Data is normalized to a median relative abundance. For a) and b), Spearman’s rank correlation coefficient is noted (r s ). The average of differences for measurements of samples A and B between competitive amplicon library preparation and TaqMan qPCR ( Figure S1 ) or Illumina RNA-sequencing ( Figure S2 ) was determined for each endogenous target; and to illustrate the systematic bias away from the regression line, data points for MMP2 have been highlighted in orange. This difference was subtracted from TaqMan qPCR or Illumina RNA-sequencing measurements for samples C and D and plotted (X-axis). Competitive amplicon library preparation measurements of C and D are plotted on the Y-axis. c) Comparison of TaqMan qPCR with competitive amplicon library preparation (n = 146) for samples C and D with correction for platform and assay specific bias. d) Comparison of Illumina RNA-Sequencing with competitive amplicon library preparation (n = 170) for samples C and D with correction for platform and assay specific bias.

    Journal: PLoS ONE

    Article Title: Targeted RNA-Sequencing with Competitive Multiplex-PCR Amplicon Libraries

    doi: 10.1371/journal.pone.0079120

    Figure Lengend Snippet: Cross-platform comparison of competitive amplicon library preparation with TaqMan qPCR and Illumina RNA-Sequencing. a ) Comparison of TaqMan qPCR with competitive amplicon library preparation (n = 146) for samples A and B without correction for systematic biases. Data is normalized to a median relative abundance. b ) Comparison of Illumina RNA-Sequencing with competitive amplicon library preparation (n = 170) for samples A and B without correction for systematic biases. Data is normalized to a median relative abundance. For a) and b), Spearman’s rank correlation coefficient is noted (r s ). The average of differences for measurements of samples A and B between competitive amplicon library preparation and TaqMan qPCR ( Figure S1 ) or Illumina RNA-sequencing ( Figure S2 ) was determined for each endogenous target; and to illustrate the systematic bias away from the regression line, data points for MMP2 have been highlighted in orange. This difference was subtracted from TaqMan qPCR or Illumina RNA-sequencing measurements for samples C and D and plotted (X-axis). Competitive amplicon library preparation measurements of C and D are plotted on the Y-axis. c) Comparison of TaqMan qPCR with competitive amplicon library preparation (n = 146) for samples C and D with correction for platform and assay specific bias. d) Comparison of Illumina RNA-Sequencing with competitive amplicon library preparation (n = 170) for samples C and D with correction for platform and assay specific bias.

    Article Snippet: We then evaluated the method for: a) accuracy and reproducibility of nucleic acid abundance measurement on different days within individual test sites, between test sites, and between different preparations of libraries, b) inter-platform concordance with TaqMan qPCR (MAQC-I study) and traditional RNA-sequencing library preparation using Illumina NGS kits (SEQC study), as well as c) reduced number of sequencing reads required for quantification.

    Techniques: Amplification, Real-time Polymerase Chain Reaction, RNA Sequencing Assay

    Length distribution and abundance of small RNAs sequences in chicken ovary by Illumina small RNA deep sequencing. Sequence length distribution of clean reads based on the abundance and distinct sequences; the most abundant size class was 22 nt, followed by 23 nt and 21 nt.

    Journal: BMC Genomics

    Article Title: Identification of miRNAs associated with sexual maturity in chicken ovary by Illumina small RNA deep sequencing

    doi: 10.1186/1471-2164-14-352

    Figure Lengend Snippet: Length distribution and abundance of small RNAs sequences in chicken ovary by Illumina small RNA deep sequencing. Sequence length distribution of clean reads based on the abundance and distinct sequences; the most abundant size class was 22 nt, followed by 23 nt and 21 nt.

    Article Snippet: The Illumina small RNA deep sequencing approach allows us to determine the relative abundance of various miRNA families by calculating the sequencing frequency.

    Techniques: Sequencing

    qRT-PCR validation of five differentially expressed miRNAs identified using Illumina small RNA deep sequencing. A . Fold-change of five miRNAs that were differentially expressed between 42-d and 162-d ovaries based on deep sequencing data. B . The relative expression abundance of the five miRNAs between 42-d and 162- d ovaries by real-time quantitative RT-PCR. * P

    Journal: BMC Genomics

    Article Title: Identification of miRNAs associated with sexual maturity in chicken ovary by Illumina small RNA deep sequencing

    doi: 10.1186/1471-2164-14-352

    Figure Lengend Snippet: qRT-PCR validation of five differentially expressed miRNAs identified using Illumina small RNA deep sequencing. A . Fold-change of five miRNAs that were differentially expressed between 42-d and 162-d ovaries based on deep sequencing data. B . The relative expression abundance of the five miRNAs between 42-d and 162- d ovaries by real-time quantitative RT-PCR. * P

    Article Snippet: The Illumina small RNA deep sequencing approach allows us to determine the relative abundance of various miRNA families by calculating the sequencing frequency.

    Techniques: Quantitative RT-PCR, Sequencing, Expressing

    ( a ) Schematic representation of the DNA oligonucleotides from which Illumina libraries were generated. ( b ) Distribution of duplex lengths amongst a sequenced sample of single-stranded DNA oligonucleotides. We extracted reads corresponding to the oligonucleotide by searching the output data for the 12 nt known sequence tag. Duplex lengths were then calculated by counting the number of bases at the 3′-end found to be the reverse complement of those at the 5′-end. This revealed a smooth distribution of values over a range of sizes, with large peaks at 12 bp (likely resulting from intermolecular annealing) and 9 bp (probably the consequence of a 3 bp duplex that can form between the known sequence tag and RNA binding site). ( c ) Two proposed mechanisms for the formation of species with 9 nt of reverse complementarity between the 5′ and 3′-ends, the most common duplex length observed. The vast majority of these were found to have 3 bp ‘seed duplexes’ formed by base pairing between –CGT– in the sequence tag and either the –GCA– in the 3′ half of the RNA oligonucleotide binding site, or the CA-dinucleotide at the start of the binding site when the preceding nucleotide (the last of the 19 nt random sequence) was G.

    Journal: Nucleic Acids Research

    Article Title: A simple method for directional transcriptome sequencing using Illumina technology

    doi: 10.1093/nar/gkp811

    Figure Lengend Snippet: ( a ) Schematic representation of the DNA oligonucleotides from which Illumina libraries were generated. ( b ) Distribution of duplex lengths amongst a sequenced sample of single-stranded DNA oligonucleotides. We extracted reads corresponding to the oligonucleotide by searching the output data for the 12 nt known sequence tag. Duplex lengths were then calculated by counting the number of bases at the 3′-end found to be the reverse complement of those at the 5′-end. This revealed a smooth distribution of values over a range of sizes, with large peaks at 12 bp (likely resulting from intermolecular annealing) and 9 bp (probably the consequence of a 3 bp duplex that can form between the known sequence tag and RNA binding site). ( c ) Two proposed mechanisms for the formation of species with 9 nt of reverse complementarity between the 5′ and 3′-ends, the most common duplex length observed. The vast majority of these were found to have 3 bp ‘seed duplexes’ formed by base pairing between –CGT– in the sequence tag and either the –GCA– in the 3′ half of the RNA oligonucleotide binding site, or the CA-dinucleotide at the start of the binding site when the preceding nucleotide (the last of the 19 nt random sequence) was G.

    Article Snippet: Illumina sequencing libraries can be generated from ss-DNA Sequencing using the Illumina platform requires the ligation of adapters, necessary for PCR amplification, flow cell attachment and sequencing reaction priming, onto either end of a DNA molecule ( ).

    Techniques: Generated, Sequencing, RNA Binding Assay, Binding Assay

    Regulation of de novo lipid synthesis in M . alpina . Pathways of de novo lipid synthesis are illustrated. Major intermediates and final products are highlighted in black, and enzymes found to be regulated at the RNA level during lipid synthesis are indicated by circled numbers. Enzymes involved in glycolysis are highlighted in light blue, pentose phosphate pathway in pink, tricarboxylic acid cycle in yellow, fatty acid synthesis in dark blue and glycerolipid synthesis in orange. Transcriptome analysis was performed in samples from 12, 2 and 0.5 h prior to and 1, 12 and 48 h after nitrogen depletion. RNA expression levels (FPKM in log 10 scale) for up-regulated enzymes are plotted in red and down-regulated enzymes in green. PK (EC 2.7.1.40): Pyruvate kinase, PCK (EC 4.1.1.32 and EC 4.1.1.49): Phosphoenolpyruvate carboxykinase, HK (EC 2.7.1.1 and EC 2.7.1.2): Hexokinase, ALDO (EC 4.1.2.13): Fructose 1,6-bisphosphate aldolase, GAPDH (EC 1.2.1.12): Glyceraldehyde 3-phosphate dehydrogenase, ENO (EC 4.2.1.11): 2-phosphoglycerate dehydratase, ACO (EC 4.2.1.3): Aconitase, IDH (EC 1.1.1.42): Isocitrate dehydrogenase, PDH (EC 1.2.4.1): Pyruvate dehydrogenase, OGDH (EC 1.2.4.2): Oxoglutarate dehydrogenase , SUCLG (EC 6.2.1.4): Succinyl-CoA synthetase, MDH (EC 1.1.1.37): Malate dehydrogenase, ACLY (EC 2.3.3.8): ATP-citric lyase, ACS (EC 6.2.1.1): Acetyl-CoA synthetase, ALDH (EC 1.2.1.3): Aldehyde dehydrogenase, ADH (EC 1.1.1.2): Alcohol dehydrogenase, GPAT (EC 2.3.1.15): 3-glycerophosphate acyltransferase, DGAT (EC 2.3.1.20): Diacylglycerol O -acyltransferase, ACSL (EC 6.2.1.3): Acyl-CoA synthetase, CRAT (EC 2.3.1.7): Carnitine O-acetyltransferase, LIP (EC 3.1.1.3): Triacylglycerol lipase, G6PD (EC 1.1.1.49): Glucose-6-phosphate dehydrogenase and PGD (EC 1.1.1.44): Phosphogluconate dehydrogenase.

    Journal: Scientific Reports

    Article Title: Identification of a critical determinant that enables efficient fatty acid synthesis in oleaginous fungi

    doi: 10.1038/srep11247

    Figure Lengend Snippet: Regulation of de novo lipid synthesis in M . alpina . Pathways of de novo lipid synthesis are illustrated. Major intermediates and final products are highlighted in black, and enzymes found to be regulated at the RNA level during lipid synthesis are indicated by circled numbers. Enzymes involved in glycolysis are highlighted in light blue, pentose phosphate pathway in pink, tricarboxylic acid cycle in yellow, fatty acid synthesis in dark blue and glycerolipid synthesis in orange. Transcriptome analysis was performed in samples from 12, 2 and 0.5 h prior to and 1, 12 and 48 h after nitrogen depletion. RNA expression levels (FPKM in log 10 scale) for up-regulated enzymes are plotted in red and down-regulated enzymes in green. PK (EC 2.7.1.40): Pyruvate kinase, PCK (EC 4.1.1.32 and EC 4.1.1.49): Phosphoenolpyruvate carboxykinase, HK (EC 2.7.1.1 and EC 2.7.1.2): Hexokinase, ALDO (EC 4.1.2.13): Fructose 1,6-bisphosphate aldolase, GAPDH (EC 1.2.1.12): Glyceraldehyde 3-phosphate dehydrogenase, ENO (EC 4.2.1.11): 2-phosphoglycerate dehydratase, ACO (EC 4.2.1.3): Aconitase, IDH (EC 1.1.1.42): Isocitrate dehydrogenase, PDH (EC 1.2.4.1): Pyruvate dehydrogenase, OGDH (EC 1.2.4.2): Oxoglutarate dehydrogenase , SUCLG (EC 6.2.1.4): Succinyl-CoA synthetase, MDH (EC 1.1.1.37): Malate dehydrogenase, ACLY (EC 2.3.3.8): ATP-citric lyase, ACS (EC 6.2.1.1): Acetyl-CoA synthetase, ALDH (EC 1.2.1.3): Aldehyde dehydrogenase, ADH (EC 1.1.1.2): Alcohol dehydrogenase, GPAT (EC 2.3.1.15): 3-glycerophosphate acyltransferase, DGAT (EC 2.3.1.20): Diacylglycerol O -acyltransferase, ACSL (EC 6.2.1.3): Acyl-CoA synthetase, CRAT (EC 2.3.1.7): Carnitine O-acetyltransferase, LIP (EC 3.1.1.3): Triacylglycerol lipase, G6PD (EC 1.1.1.49): Glucose-6-phosphate dehydrogenase and PGD (EC 1.1.1.44): Phosphogluconate dehydrogenase.

    Article Snippet: Transcriptome analysis Paired RNA-sequencing reads of 100 bp in length were obtained from the Illumina GA IIx sequencing platform, and all of the raw data were deposited in Sequence Read Archive database ( http://www.ncbi.nlm.nih.gov/Traces/sra/ ) under the accession numbers of SRR1638088, SRR1638089, SRR1638091, SRR1638092, SRR1638093, and SRR1638095.

    Techniques: RNA Expression