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Illumina Inc cdna library construction
Schematic of <t>Illumina</t> EST analysis. It includes sample preparation, <t>cDNA</t> library construction and Illumina sequencing, data analysis including assemble, blast, GO annotation, gene expression analysis, etc.
Cdna Library Construction, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 143 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Deep Sequencing-Based Transcriptome Analysis of Chicken Spleen in Response to Avian Pathogenic Escherichia coli (APEC) Infection"

Article Title: Deep Sequencing-Based Transcriptome Analysis of Chicken Spleen in Response to Avian Pathogenic Escherichia coli (APEC) Infection

Journal: PLoS ONE

doi: 10.1371/journal.pone.0041645

Schematic of Illumina EST analysis. It includes sample preparation, cDNA library construction and Illumina sequencing, data analysis including assemble, blast, GO annotation, gene expression analysis, etc.
Figure Legend Snippet: Schematic of Illumina EST analysis. It includes sample preparation, cDNA library construction and Illumina sequencing, data analysis including assemble, blast, GO annotation, gene expression analysis, etc.

Techniques Used: Sample Prep, cDNA Library Assay, Sequencing, Expressing

Related Articles

Amplification:

Article Title: The Complete Campylobacter jejuni Transcriptome during Colonization of a Natural Host Determined by RNAseq
Article Snippet: Conformation of DNA removal was assessed by PCR with three sets of primers ranging in amplification products of 100, 150, 250 bp and the Qubit dsDNA high sensitivity assay kit (Life Technologies) (data not shown). .. Purified DNA-free RNA from 5 birds (housed in the same brooder) was pooled to create enough starting DNA-free RNA for Illumina cDNA library construction.

Synthesized:

Article Title: The draft genome assembly of Rhododendron delavayi Franch. var. delavayi
Article Snippet: Random oligo-nucleotides and M-MuLV Reverse Transcriptase (RNase H) were used to synthesize the first cDNA strand, and then the second cDNA strand was synthesized using DNA Polymerase I and RNase H. The cDNA libraries with insert sizes of 200–500 base pairs (bps) were selected and purified with the AMPure XP beads system (Beckman Coulter), and subsequently sequenced on an Illumina HiSeq 2000 platform. .. Both cDNA library construction and Illumina sequencing were carried out by BGI-ShenZhen.

Construct:

Article Title: The gut microbiome in atherosclerotic cardiovascular disease
Article Snippet: DNA library construction was performed following the manufacturer’s instruction (Illumina). .. We used the same workflow as described previously to perform cluster generation, template hybridization, isothermal amplification, linearization, blocking and denaturation, and hybridization of the sequencing primers.

Article Title: Impact of a 3-Months Vegetarian Diet on the Gut Microbiota and Immune Repertoire
Article Snippet: DNA library construction was performed following the manufacturer’s instruction (Illumina). .. A similar workflow as described previously ( ) was used to perform cluster generation, template hybridization, isothermal amplification, linearization, blocking and denaturation, and hybridization of the sequencing primers.

Article Title: The draft genome assembly of Rhododendron delavayi Franch. var. delavayi
Article Snippet: A total of 336.83 Gb (×482.61) raw reads were generated from all constructed libraries. .. Both cDNA library construction and Illumina sequencing were carried out by BGI-ShenZhen.

Incubation:

Article Title: Leptin Stimulates Cellular Glycolysis Through a STAT3 Dependent Mechanism in Tilapia
Article Snippet: For RNA-seq studies assessing leptin modulation of the pituitary RPD transcriptome, tissues were placed in wells of a Falcon 6-well plate in 3 mL of either hormone-free (control) or leptin-containing medium and incubated for 6 h (n = 30/treatment; 10 RPD/well, 3 wells/treatment). .. Pituitary mRNA (10 μg) was submitted to the North Carolina State University Genomic Sciences Laboratory (Raleigh, NC) for Illumina cDNA library construction.

Formalin-fixed Paraffin-Embedded:

Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA
Article Snippet: The data presented demonstrate that the immobilized enzymes protocol can be applied to ideal genomic samples with plenty of material recently extracted. .. To further assess the utility of the immobilized enzymes method on more challenging samples, we have performed Illumina DNA library construction using human FFPE DNA with or without DNA repair as described previously . .. We used FFPE DNA samples as an example of challenging genomic DNA for which library preparation has been shown to be problematic and DNA has been shown to be extensively damaged, notably cytosine deamination .

RNA Detection:

Article Title: Transcriptomic profiling of Melilotus albus near-isogenic lines contrasting for coumarin content
Article Snippet: For each genotype, high-quality RNA from three replications was used for cDNA library construction and Illumina deep sequencing. .. Fifteen cDNA libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA).

Gas Chromatography:

Article Title: De Novo Assembly and Analysis of Tartary Buckwheat (Fagopyrum tataricum Garetn.) Transcriptome Discloses Key Regulators Involved in Salt-Stress Response
Article Snippet: Total RNA of control and salt treated samples were extracted, followed by cDNA library construction and paired-end Illumina deep sequencing. .. Total RNA of control and salt treated samples were extracted, followed by cDNA library construction and paired-end Illumina deep sequencing.

Biomarker Assay:

Article Title: Differential transcriptome analysis reveals genes related to cold tolerance in seabuckthorn carpenter moth, Eogystia hippophaecolus
Article Snippet: The mRNA samples were purified and fragmented using the TruSeq RNA Sample Preparation Kit v2-Set A (No. RS-122-2001; Illumina, San Diego, CA, USA) to remove rRNA. .. After additional quality control, cDNA library construction, Illumina sequencing, and de novo transcriptome assembly were performed by the Beijing Biomarker Biotechnology Co. (Beijing, China). .. In view of the costs, 5°C and -5°C samples were pooled by instar to construct the cDNA library.

Cell Culture:

Article Title: Comparative transcript profiling of resistant and susceptible peanut post-harvest seeds in response to aflatoxin production by Aspergillus flavus
Article Snippet: Seeds of the R and S genotypes inoculated with A. flavus (treatment) and without inoculation (control) cultured for 1, 3 and 7 days were sampled for RNA isolation and cDNA library construction. .. RNA quality detection, cDNA library construction, and Illumina deep sequencing were performed following the previous method [ , – ].

Reverse Transcription Polymerase Chain Reaction:

Article Title: Complete Genome Sequences of Two Dengue Virus Serotype 1 Genotype V Strains from Different Lineages
Article Snippet: DENV1 was detected by multiplex reverse transcription-PCR (RT-PCR), using as a strategy cDNA production by Flavivirus -gender-specific primers ( ). .. After positive DENV1 detections, the DNA was transcribed again, now using random primers for DNA library construction and Illumina genome sequencing ( ).

Generated:

Article Title: Transcriptomic profiling of Melilotus albus near-isogenic lines contrasting for coumarin content
Article Snippet: For each genotype, high-quality RNA from three replications was used for cDNA library construction and Illumina deep sequencing. .. For each genotype, high-quality RNA from three replications was used for cDNA library construction and Illumina deep sequencing.

Article Title: De Novo Assembly and Analysis of Tartary Buckwheat (Fagopyrum tataricum Garetn.) Transcriptome Discloses Key Regulators Involved in Salt-Stress Response
Article Snippet: Total RNA of control and salt treated samples were extracted, followed by cDNA library construction and paired-end Illumina deep sequencing. .. Total RNA of control and salt treated samples were extracted, followed by cDNA library construction and paired-end Illumina deep sequencing.

Article Title: The draft genome assembly of Rhododendron delavayi Franch. var. delavayi
Article Snippet: A total of 336.83 Gb (×482.61) raw reads were generated from all constructed libraries. .. Both cDNA library construction and Illumina sequencing were carried out by BGI-ShenZhen.

Article Title: RNA-Seq Uncovers SNPs and Alternative Splicing Events in Asian Lotus (Nelumbo nucifera)
Article Snippet: These mixed RNA samples were subsequently used in cDNA library construction and Illumina sequencing. .. A total amount of 3 μg RNA per sample was used to construct cDNA library.

Sequencing:

Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA
Article Snippet: To further assess the utility of the immobilized enzymes method on more challenging samples, we have performed Illumina DNA library construction using human FFPE DNA with or without DNA repair as described previously . .. We used FFPE DNA samples as an example of challenging genomic DNA for which library preparation has been shown to be problematic and DNA has been shown to be extensively damaged, notably cytosine deamination .

Article Title: Deep Sequencing-Based Transcriptome Analysis of Chicken Spleen in Response to Avian Pathogenic Escherichia coli (APEC) Infection
Article Snippet: Then, samples of three individuals were pooled within each group in equal amounts to generate one mixed sample per group by RNA pooling. .. These three mixed RNA samples were subsequently used in cDNA library construction and Illumina deep sequencing. .. Three cDNA libraries were prepared using the TruseqTM RNA sample prep Kit (Illumina, San Diego, CA USA) following the manufacturer’s instructions.

Article Title: Leptin Stimulates Cellular Glycolysis Through a STAT3 Dependent Mechanism in Tilapia
Article Snippet: Pituitary mRNA (10 μg) was submitted to the North Carolina State University Genomic Sciences Laboratory (Raleigh, NC) for Illumina cDNA library construction. .. Pituitary mRNA (10 μg) was submitted to the North Carolina State University Genomic Sciences Laboratory (Raleigh, NC) for Illumina cDNA library construction.

Article Title: The gut microbiome in atherosclerotic cardiovascular disease
Article Snippet: Paragraph title: DNA library construction and sequencing of fecal samples ... DNA library construction was performed following the manufacturer’s instruction (Illumina).

Article Title: Transient O2 pulses direct Fe crystallinity and Fe(III)-reducer gene expression within a soil microbiome
Article Snippet: Gives Mössbauer parameters for spectra recorded at 295 K. (XLSX 47 kb) .. We thank Whendee Silver (UC Berkeley) and Steven Hall (U Iowa) for providing access to the Bisley site; Nehru Mantripragada (Crop and Soil Science Dept., University of Georgia (UGA)) for the laboratory assistance; Edward T. Kipreos (Dept. of Cellular Biology, UGA) for the use of laboratory space and equipment; and Katherine Sandlin and Roger Nilsen of the Georgia Genomics and Bioinformatics Core (GGBC), Athens, GA for cDNA library construction, Illumina sequencing, and consultation. .. We also acknowledge the Georgia Advanced Computing Resource Center (GACRC) at UGA for the Linux system use during the meta-data handling and processing.

Article Title: Differential transcriptome analysis reveals genes related to cold tolerance in seabuckthorn carpenter moth, Eogystia hippophaecolus
Article Snippet: The mRNA samples were purified and fragmented using the TruSeq RNA Sample Preparation Kit v2-Set A (No. RS-122-2001; Illumina, San Diego, CA, USA) to remove rRNA. .. After additional quality control, cDNA library construction, Illumina sequencing, and de novo transcriptome assembly were performed by the Beijing Biomarker Biotechnology Co. (Beijing, China). .. In view of the costs, 5°C and -5°C samples were pooled by instar to construct the cDNA library.

Article Title: Comparative transcriptomic analysis reveals the roles of ROS scavenging genes in response to cadmium in two pak choi cultivars
Article Snippet: Total RNA was extracted using Trizol® Reagent (Invitrogen) from a total of eight individual samples. .. Approximately 1.5 µg of the extracted RNA per sample was used as input material for cDNA library construction and subsequent Illumina sequencing. .. The eight cDNA libraries (B1_Cd0 , B2_Cd0 , B1_Cd10 , B2_Cd10 , K1_Cd0 , K2_Cd0 , K1_Cd10 and K2_Cd10 ) were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer’s instructions : (i) mRNA was purified using poly-T oligo-attached magnetic beads; (ii) fragmentation was carried out using divalent cations under elevated temperature in NEBNext; (iii) first strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H− ); (iv) after second strand cDNA synthesis and adaptor ligation, the cDNA fragments were isolated and purified with AMPure XP system (Beckman Coulter, Beverly, USA); (v) Then PCR amplifications were selected as templates to create the final cDNA library, and library quality was assessed on the Agilent Bioanalyzer 2100 system.

Article Title: Transcriptomic profiling of Melilotus albus near-isogenic lines contrasting for coumarin content
Article Snippet: The quality and quantity of total RNA were analyzed using an UltrasecTM 2100 pro UV/Visible Spectrophotometer (Amersham Biosciences, Uppsala, Sweden) and by gel electrophoresis. .. For each genotype, high-quality RNA from three replications was used for cDNA library construction and Illumina deep sequencing. .. A total amount of 3 µg RNA per sample was used as input material for the RNA sample preparations.

Article Title: De Novo Assembly and Analysis of Tartary Buckwheat (Fagopyrum tataricum Garetn.) Transcriptome Discloses Key Regulators Involved in Salt-Stress Response
Article Snippet: To elucidate the underlying salt stress responsive mechanisms in tartary buckwheat, we carried out RNA-seq analysis. .. Total RNA of control and salt treated samples were extracted, followed by cDNA library construction and paired-end Illumina deep sequencing. .. After removing the low-quality reads and adaptor sequences from reads, in total we obtain 23.81 million and 29.34 million clean reads from control and salt treatment libraries, which yielded 7.14 billion and 8.80 billion nucleotides, respectively ( ).

Article Title: Impact of a 3-Months Vegetarian Diet on the Gut Microbiota and Immune Repertoire
Article Snippet: Paragraph title: DNA Library Construction and Metagenome Sequencing ... DNA library construction was performed following the manufacturer’s instruction (Illumina).

Article Title: Comparative transcript profiling of resistant and susceptible peanut post-harvest seeds in response to aflatoxin production by Aspergillus flavus
Article Snippet: The concentration and integrity of the pooled total RNA was checked using a NanoDrop® 2000 spectrophotometer, a Qubit® Fluorometer 2.0, and an Agilent 2100 bioanalyzer, to confirm that all samples had an RNA integrity number greater than 6.5. .. RNA quality detection, cDNA library construction, and Illumina deep sequencing were performed following the previous method [ , – ]. .. Raw data (raw reads) in the fastq format were first processed using in-house perl scripts.

Article Title: Regulation of malonyl-CoA-acyl carrier protein transacylase network in umbilical cord blood affected by intrauterine hyperglycemia
Article Snippet: Total RNA was dissolved in RNase-free water and RNA integrity was measured using Agilent 2100 bioanalyzer (Quantifluor-ST fluorometer, Promega, E6090). .. The high quality RNA was used for cDNA library construction and Illumina sequencing. .. Poly (A) mRNA was isolated from the total RNA using the PolyA+ Tract mRNA Isolation System (Illumina, San Diego, CA), and further purified using the RNeasy MinElute Clean up Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol.

Article Title: The draft genome assembly of Rhododendron delavayi Franch. var. delavayi
Article Snippet: Random oligo-nucleotides and M-MuLV Reverse Transcriptase (RNase H) were used to synthesize the first cDNA strand, and then the second cDNA strand was synthesized using DNA Polymerase I and RNase H. The cDNA libraries with insert sizes of 200–500 base pairs (bps) were selected and purified with the AMPure XP beads system (Beckman Coulter), and subsequently sequenced on an Illumina HiSeq 2000 platform. .. Both cDNA library construction and Illumina sequencing were carried out by BGI-ShenZhen. .. Paired-end reads were generated with a read length of 90 bp.

Article Title: Complete Genome Sequences of Two Dengue Virus Serotype 1 Genotype V Strains from Different Lineages
Article Snippet: DENV1 was detected by multiplex reverse transcription-PCR (RT-PCR), using as a strategy cDNA production by Flavivirus -gender-specific primers ( ). .. After positive DENV1 detections, the DNA was transcribed again, now using random primers for DNA library construction and Illumina genome sequencing ( ). .. For this project, 11 complete genomes were sequenced, with two of them described in this announcement, those of strains BR/SJRP/287/2011 and BR/SJRP/287/2011.

Article Title: RNA-Seq Uncovers SNPs and Alternative Splicing Events in Asian Lotus (Nelumbo nucifera)
Article Snippet: For each developmental stage of the four cultivars, the RNA samples from the three individual repeats were pooled together in equal amounts to generate one mixed sample. .. These mixed RNA samples were subsequently used in cDNA library construction and Illumina sequencing. .. A total amount of 3 μg RNA per sample was used to construct cDNA library.

Article Title: De Novo Analysis Reveals Transcriptomic Responses in Eriobotrya japonica Fruits during Postharvest Cold Storage
Article Snippet: The predicted starch and sucrose metabolism pathway was drawn by Powerpoint software (Microsoft, Redmond, WA, USA). .. Total RNA of cold stored loquat fruit pulps was extracted and eligible RNA samples ( ) were used to cDNA library construction and Illumina transcriptome sequencing. .. In total, 2,289,564,890 raw reads were generated from eighteen cDNA libraries and 2,207,813,200 clean reads were obtained by filtering adapters, unknown nucleotides and low-quality reads ( ).

Nucleic Acid Electrophoresis:

Article Title: Transcriptomic profiling of Melilotus albus near-isogenic lines contrasting for coumarin content
Article Snippet: The quality and quantity of total RNA were analyzed using an UltrasecTM 2100 pro UV/Visible Spectrophotometer (Amersham Biosciences, Uppsala, Sweden) and by gel electrophoresis. .. For each genotype, high-quality RNA from three replications was used for cDNA library construction and Illumina deep sequencing.

In Vivo:

Article Title: The Complete Campylobacter jejuni Transcriptome during Colonization of a Natural Host Determined by RNAseq
Article Snippet: For in vivo RNA isolation, cecal contents were harvested from individual birds and immediately submerged in 1 ml of RNAlater/cecum. .. Purified DNA-free RNA from 5 birds (housed in the same brooder) was pooled to create enough starting DNA-free RNA for Illumina cDNA library construction.

RNA Sequencing Assay:

Article Title: Leptin Stimulates Cellular Glycolysis Through a STAT3 Dependent Mechanism in Tilapia
Article Snippet: Paragraph title: RNA-seq and bioinformatic analyses of the pituitary transcriptome ... Pituitary mRNA (10 μg) was submitted to the North Carolina State University Genomic Sciences Laboratory (Raleigh, NC) for Illumina cDNA library construction.

Article Title: De Novo Assembly and Analysis of Tartary Buckwheat (Fagopyrum tataricum Garetn.) Transcriptome Discloses Key Regulators Involved in Salt-Stress Response
Article Snippet: Paragraph title: 3.2. RNA-Seq and De Novo Assembly of the F. tataricum Transcriptome ... Total RNA of control and salt treated samples were extracted, followed by cDNA library construction and paired-end Illumina deep sequencing.

Article Title: Unraveling multifaceted contributions of small regulatory RNAs to photomorphogenic development in Arabidopsis
Article Snippet: Paragraph title: RNA sequencing and data analyses ... Ten to 15 μg total RNA was size fractionated on 15% Tris-Borate-EDTA-Urea gel. sRNAs ranging from 17 to 30 nucleotides were gel-purified and used for cDNA library construction (Illumina Truseq for replicates 1 and 2, Small RNA v1.5 for replicates 3) and sequencing with the use of an Illumina HiSeq 2500.

Sensitive Assay:

Article Title: The Complete Campylobacter jejuni Transcriptome during Colonization of a Natural Host Determined by RNAseq
Article Snippet: Conformation of DNA removal was assessed by PCR with three sets of primers ranging in amplification products of 100, 150, 250 bp and the Qubit dsDNA high sensitivity assay kit (Life Technologies) (data not shown). .. Purified DNA-free RNA from 5 birds (housed in the same brooder) was pooled to create enough starting DNA-free RNA for Illumina cDNA library construction.

Magnetic Beads:

Article Title: Deep Sequencing-Based Transcriptome Analysis of Chicken Spleen in Response to Avian Pathogenic Escherichia coli (APEC) Infection
Article Snippet: These three mixed RNA samples were subsequently used in cDNA library construction and Illumina deep sequencing. .. Three cDNA libraries were prepared using the TruseqTM RNA sample prep Kit (Illumina, San Diego, CA USA) following the manufacturer’s instructions.

Article Title: RNA-Seq Uncovers SNPs and Alternative Splicing Events in Asian Lotus (Nelumbo nucifera)
Article Snippet: These mixed RNA samples were subsequently used in cDNA library construction and Illumina sequencing. .. The library was generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer’s recommendations.

Isolation:

Article Title: Deep Sequencing-Based Transcriptome Analysis of Chicken Spleen in Response to Avian Pathogenic Escherichia coli (APEC) Infection
Article Snippet: Paragraph title: RNA Isolation, cDNA Library Construction and Illumina Deep Sequencing ... These three mixed RNA samples were subsequently used in cDNA library construction and Illumina deep sequencing.

Article Title: Leptin Stimulates Cellular Glycolysis Through a STAT3 Dependent Mechanism in Tilapia
Article Snippet: Tissues from each well were pooled in 2 mL of RNAlater at 4°C until RNA isolation and Illumina cDNA library preparation. .. Pituitary mRNA (10 μg) was submitted to the North Carolina State University Genomic Sciences Laboratory (Raleigh, NC) for Illumina cDNA library construction.

Article Title: Comparative transcriptomic analysis reveals the roles of ROS scavenging genes in response to cadmium in two pak choi cultivars
Article Snippet: Paragraph title: RNA isolation, cDNA library construction and sequencing ... Approximately 1.5 µg of the extracted RNA per sample was used as input material for cDNA library construction and subsequent Illumina sequencing.

Article Title: Transcriptomic profiling of Melilotus albus near-isogenic lines contrasting for coumarin content
Article Snippet: Paragraph title: RNA isolation and library preparation for transcriptome analysis ... For each genotype, high-quality RNA from three replications was used for cDNA library construction and Illumina deep sequencing.

Article Title: Comparative transcript profiling of resistant and susceptible peanut post-harvest seeds in response to aflatoxin production by Aspergillus flavus
Article Snippet: Paragraph title: RNA isolation and cDNA library construction ... RNA quality detection, cDNA library construction, and Illumina deep sequencing were performed following the previous method [ , – ].

Article Title: The Complete Campylobacter jejuni Transcriptome during Colonization of a Natural Host Determined by RNAseq
Article Snippet: Paragraph title: RNA isolation ... Purified DNA-free RNA from 5 birds (housed in the same brooder) was pooled to create enough starting DNA-free RNA for Illumina cDNA library construction.

Article Title: RNA-Seq Uncovers SNPs and Alternative Splicing Events in Asian Lotus (Nelumbo nucifera)
Article Snippet: Paragraph title: RNA isolation, library construction, and sequencing ... These mixed RNA samples were subsequently used in cDNA library construction and Illumina sequencing.

Article Title: Unraveling multifaceted contributions of small regulatory RNAs to photomorphogenic development in Arabidopsis
Article Snippet: For sRNA sequencing and data analyses, 4-d-old dark-grown Arabidopsis Col-0 seedlings were exposed to white light (100 μE) for 1, 3, 6, 12 and 24 h. The aerial tissues of approximately 5000 seedlings were collected for RNA isolation. .. Ten to 15 μg total RNA was size fractionated on 15% Tris-Borate-EDTA-Urea gel. sRNAs ranging from 17 to 30 nucleotides were gel-purified and used for cDNA library construction (Illumina Truseq for replicates 1 and 2, Small RNA v1.5 for replicates 3) and sequencing with the use of an Illumina HiSeq 2500.

RNA Extraction:

Article Title: Differential transcriptome analysis reveals genes related to cold tolerance in seabuckthorn carpenter moth, Eogystia hippophaecolus
Article Snippet: Paragraph title: Insect material and RNA extraction ... After additional quality control, cDNA library construction, Illumina sequencing, and de novo transcriptome assembly were performed by the Beijing Biomarker Biotechnology Co. (Beijing, China).

Article Title: Regulation of malonyl-CoA-acyl carrier protein transacylase network in umbilical cord blood affected by intrauterine hyperglycemia
Article Snippet: Total RNA was extracted using RNAiso Plus Total RNA extraction reagent (TaKaRa, Shiga, Japan) following the manufacturer’s instructions. .. The high quality RNA was used for cDNA library construction and Illumina sequencing.

Purification:

Article Title: Differential transcriptome analysis reveals genes related to cold tolerance in seabuckthorn carpenter moth, Eogystia hippophaecolus
Article Snippet: The mRNA samples were purified and fragmented using the TruSeq RNA Sample Preparation Kit v2-Set A (No. RS-122-2001; Illumina, San Diego, CA, USA) to remove rRNA. .. After additional quality control, cDNA library construction, Illumina sequencing, and de novo transcriptome assembly were performed by the Beijing Biomarker Biotechnology Co. (Beijing, China).

Article Title: Transcriptomic profiling of Melilotus albus near-isogenic lines contrasting for coumarin content
Article Snippet: Total RNA of six leaves from three samples in each genotype was isolated as three biological replications using an RNAprep pure Plant RNA Purification Kit (Tiangen Biotech, Beijing, China). .. For each genotype, high-quality RNA from three replications was used for cDNA library construction and Illumina deep sequencing.

Article Title: The Complete Campylobacter jejuni Transcriptome during Colonization of a Natural Host Determined by RNAseq
Article Snippet: After C. jejuni enrichment, RNA was extracted with TRIzol® and contaminating DNA was removed with TURBO™ DNase as described previously. .. Purified DNA-free RNA from 5 birds (housed in the same brooder) was pooled to create enough starting DNA-free RNA for Illumina cDNA library construction. .. Illumina cDNA libraries were constructed in a similar manner to Mandlik et al. [ ].

Article Title: The draft genome assembly of Rhododendron delavayi Franch. var. delavayi
Article Snippet: Random oligo-nucleotides and M-MuLV Reverse Transcriptase (RNase H) were used to synthesize the first cDNA strand, and then the second cDNA strand was synthesized using DNA Polymerase I and RNase H. The cDNA libraries with insert sizes of 200–500 base pairs (bps) were selected and purified with the AMPure XP beads system (Beckman Coulter), and subsequently sequenced on an Illumina HiSeq 2000 platform. .. Both cDNA library construction and Illumina sequencing were carried out by BGI-ShenZhen.

Article Title: RNA-Seq Uncovers SNPs and Alternative Splicing Events in Asian Lotus (Nelumbo nucifera)
Article Snippet: These mixed RNA samples were subsequently used in cDNA library construction and Illumina sequencing. .. The library was generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer’s recommendations.

Polymerase Chain Reaction:

Article Title: The Complete Campylobacter jejuni Transcriptome during Colonization of a Natural Host Determined by RNAseq
Article Snippet: Conformation of DNA removal was assessed by PCR with three sets of primers ranging in amplification products of 100, 150, 250 bp and the Qubit dsDNA high sensitivity assay kit (Life Technologies) (data not shown). .. Purified DNA-free RNA from 5 birds (housed in the same brooder) was pooled to create enough starting DNA-free RNA for Illumina cDNA library construction.

Article Title: The draft genome assembly of Rhododendron delavayi Franch. var. delavayi
Article Snippet: Before assembly, reads with low-quality polymerase chain reaction duplication and adapter contaminations were filtered by SOAPfilter, as included in SOAPdenovo v. 2.04 (SOAPdenovo2, RRID:SCR_014986 ) [ ], and finally 246.06 Gb (×352.55) high-quality sequences were obtained for genome assembly. .. Both cDNA library construction and Illumina sequencing were carried out by BGI-ShenZhen.

cDNA Library Assay:

Article Title: Deep Sequencing-Based Transcriptome Analysis of Chicken Spleen in Response to Avian Pathogenic Escherichia coli (APEC) Infection
Article Snippet: Then, samples of three individuals were pooled within each group in equal amounts to generate one mixed sample per group by RNA pooling. .. These three mixed RNA samples were subsequently used in cDNA library construction and Illumina deep sequencing. .. Three cDNA libraries were prepared using the TruseqTM RNA sample prep Kit (Illumina, San Diego, CA USA) following the manufacturer’s instructions.

Article Title: Leptin Stimulates Cellular Glycolysis Through a STAT3 Dependent Mechanism in Tilapia
Article Snippet: Tissues from each well were pooled in 2 mL of RNAlater at 4°C until RNA isolation and Illumina cDNA library preparation. .. Pituitary mRNA (10 μg) was submitted to the North Carolina State University Genomic Sciences Laboratory (Raleigh, NC) for Illumina cDNA library construction. .. The cDNA was synthesized using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen) and tagged with a 5′, four-nucleotide barcode.

Article Title: Transient O2 pulses direct Fe crystallinity and Fe(III)-reducer gene expression within a soil microbiome
Article Snippet: Gives Mössbauer parameters for spectra recorded at 295 K. (XLSX 47 kb) .. We thank Whendee Silver (UC Berkeley) and Steven Hall (U Iowa) for providing access to the Bisley site; Nehru Mantripragada (Crop and Soil Science Dept., University of Georgia (UGA)) for the laboratory assistance; Edward T. Kipreos (Dept. of Cellular Biology, UGA) for the use of laboratory space and equipment; and Katherine Sandlin and Roger Nilsen of the Georgia Genomics and Bioinformatics Core (GGBC), Athens, GA for cDNA library construction, Illumina sequencing, and consultation. .. We also acknowledge the Georgia Advanced Computing Resource Center (GACRC) at UGA for the Linux system use during the meta-data handling and processing.

Article Title: Differential transcriptome analysis reveals genes related to cold tolerance in seabuckthorn carpenter moth, Eogystia hippophaecolus
Article Snippet: The mRNA samples were purified and fragmented using the TruSeq RNA Sample Preparation Kit v2-Set A (No. RS-122-2001; Illumina, San Diego, CA, USA) to remove rRNA. .. After additional quality control, cDNA library construction, Illumina sequencing, and de novo transcriptome assembly were performed by the Beijing Biomarker Biotechnology Co. (Beijing, China). .. In view of the costs, 5°C and -5°C samples were pooled by instar to construct the cDNA library.

Article Title: Comparative transcriptomic analysis reveals the roles of ROS scavenging genes in response to cadmium in two pak choi cultivars
Article Snippet: Total RNA was extracted using Trizol® Reagent (Invitrogen) from a total of eight individual samples. .. Approximately 1.5 µg of the extracted RNA per sample was used as input material for cDNA library construction and subsequent Illumina sequencing. .. The eight cDNA libraries (B1_Cd0 , B2_Cd0 , B1_Cd10 , B2_Cd10 , K1_Cd0 , K2_Cd0 , K1_Cd10 and K2_Cd10 ) were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer’s instructions : (i) mRNA was purified using poly-T oligo-attached magnetic beads; (ii) fragmentation was carried out using divalent cations under elevated temperature in NEBNext; (iii) first strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H− ); (iv) after second strand cDNA synthesis and adaptor ligation, the cDNA fragments were isolated and purified with AMPure XP system (Beckman Coulter, Beverly, USA); (v) Then PCR amplifications were selected as templates to create the final cDNA library, and library quality was assessed on the Agilent Bioanalyzer 2100 system.

Article Title: Transcriptomic profiling of Melilotus albus near-isogenic lines contrasting for coumarin content
Article Snippet: The quality and quantity of total RNA were analyzed using an UltrasecTM 2100 pro UV/Visible Spectrophotometer (Amersham Biosciences, Uppsala, Sweden) and by gel electrophoresis. .. For each genotype, high-quality RNA from three replications was used for cDNA library construction and Illumina deep sequencing. .. A total amount of 3 µg RNA per sample was used as input material for the RNA sample preparations.

Article Title: De Novo Assembly and Analysis of Tartary Buckwheat (Fagopyrum tataricum Garetn.) Transcriptome Discloses Key Regulators Involved in Salt-Stress Response
Article Snippet: To elucidate the underlying salt stress responsive mechanisms in tartary buckwheat, we carried out RNA-seq analysis. .. Total RNA of control and salt treated samples were extracted, followed by cDNA library construction and paired-end Illumina deep sequencing. .. After removing the low-quality reads and adaptor sequences from reads, in total we obtain 23.81 million and 29.34 million clean reads from control and salt treatment libraries, which yielded 7.14 billion and 8.80 billion nucleotides, respectively ( ).

Article Title: Comparative transcript profiling of resistant and susceptible peanut post-harvest seeds in response to aflatoxin production by Aspergillus flavus
Article Snippet: The concentration and integrity of the pooled total RNA was checked using a NanoDrop® 2000 spectrophotometer, a Qubit® Fluorometer 2.0, and an Agilent 2100 bioanalyzer, to confirm that all samples had an RNA integrity number greater than 6.5. .. RNA quality detection, cDNA library construction, and Illumina deep sequencing were performed following the previous method [ , – ]. .. Raw data (raw reads) in the fastq format were first processed using in-house perl scripts.

Article Title: Regulation of malonyl-CoA-acyl carrier protein transacylase network in umbilical cord blood affected by intrauterine hyperglycemia
Article Snippet: Total RNA was dissolved in RNase-free water and RNA integrity was measured using Agilent 2100 bioanalyzer (Quantifluor-ST fluorometer, Promega, E6090). .. The high quality RNA was used for cDNA library construction and Illumina sequencing. .. Poly (A) mRNA was isolated from the total RNA using the PolyA+ Tract mRNA Isolation System (Illumina, San Diego, CA), and further purified using the RNeasy MinElute Clean up Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol.

Article Title: The Complete Campylobacter jejuni Transcriptome during Colonization of a Natural Host Determined by RNAseq
Article Snippet: After C. jejuni enrichment, RNA was extracted with TRIzol® and contaminating DNA was removed with TURBO™ DNase as described previously. .. Purified DNA-free RNA from 5 birds (housed in the same brooder) was pooled to create enough starting DNA-free RNA for Illumina cDNA library construction. .. Illumina cDNA libraries were constructed in a similar manner to Mandlik et al. [ ].

Article Title: The draft genome assembly of Rhododendron delavayi Franch. var. delavayi
Article Snippet: Random oligo-nucleotides and M-MuLV Reverse Transcriptase (RNase H) were used to synthesize the first cDNA strand, and then the second cDNA strand was synthesized using DNA Polymerase I and RNase H. The cDNA libraries with insert sizes of 200–500 base pairs (bps) were selected and purified with the AMPure XP beads system (Beckman Coulter), and subsequently sequenced on an Illumina HiSeq 2000 platform. .. Both cDNA library construction and Illumina sequencing were carried out by BGI-ShenZhen. .. Paired-end reads were generated with a read length of 90 bp.

Article Title: RNA-Seq Uncovers SNPs and Alternative Splicing Events in Asian Lotus (Nelumbo nucifera)
Article Snippet: For each developmental stage of the four cultivars, the RNA samples from the three individual repeats were pooled together in equal amounts to generate one mixed sample. .. These mixed RNA samples were subsequently used in cDNA library construction and Illumina sequencing. .. A total amount of 3 μg RNA per sample was used to construct cDNA library.

Article Title: De Novo Analysis Reveals Transcriptomic Responses in Eriobotrya japonica Fruits during Postharvest Cold Storage
Article Snippet: The predicted starch and sucrose metabolism pathway was drawn by Powerpoint software (Microsoft, Redmond, WA, USA). .. Total RNA of cold stored loquat fruit pulps was extracted and eligible RNA samples ( ) were used to cDNA library construction and Illumina transcriptome sequencing. .. In total, 2,289,564,890 raw reads were generated from eighteen cDNA libraries and 2,207,813,200 clean reads were obtained by filtering adapters, unknown nucleotides and low-quality reads ( ).

Sample Prep:

Article Title: Differential transcriptome analysis reveals genes related to cold tolerance in seabuckthorn carpenter moth, Eogystia hippophaecolus
Article Snippet: The mRNA samples were purified and fragmented using the TruSeq RNA Sample Preparation Kit v2-Set A (No. RS-122-2001; Illumina, San Diego, CA, USA) to remove rRNA. .. After additional quality control, cDNA library construction, Illumina sequencing, and de novo transcriptome assembly were performed by the Beijing Biomarker Biotechnology Co. (Beijing, China).

Multiplex Assay:

Article Title: Complete Genome Sequences of Two Dengue Virus Serotype 1 Genotype V Strains from Different Lineages
Article Snippet: DENV1 was detected by multiplex reverse transcription-PCR (RT-PCR), using as a strategy cDNA production by Flavivirus -gender-specific primers ( ). .. After positive DENV1 detections, the DNA was transcribed again, now using random primers for DNA library construction and Illumina genome sequencing ( ).

Agarose Gel Electrophoresis:

Article Title: RNA-Seq Uncovers SNPs and Alternative Splicing Events in Asian Lotus (Nelumbo nucifera)
Article Snippet: RNA integrity was evaluated with a 1.0% agarose gel stained with ethidium bromide (EB). .. These mixed RNA samples were subsequently used in cDNA library construction and Illumina sequencing.

Spectrophotometry:

Article Title: Transcriptomic profiling of Melilotus albus near-isogenic lines contrasting for coumarin content
Article Snippet: The quality and quantity of total RNA were analyzed using an UltrasecTM 2100 pro UV/Visible Spectrophotometer (Amersham Biosciences, Uppsala, Sweden) and by gel electrophoresis. .. For each genotype, high-quality RNA from three replications was used for cDNA library construction and Illumina deep sequencing.

Article Title: Comparative transcript profiling of resistant and susceptible peanut post-harvest seeds in response to aflatoxin production by Aspergillus flavus
Article Snippet: The concentration and integrity of the pooled total RNA was checked using a NanoDrop® 2000 spectrophotometer, a Qubit® Fluorometer 2.0, and an Agilent 2100 bioanalyzer, to confirm that all samples had an RNA integrity number greater than 6.5. .. RNA quality detection, cDNA library construction, and Illumina deep sequencing were performed following the previous method [ , – ].

Article Title: RNA-Seq Uncovers SNPs and Alternative Splicing Events in Asian Lotus (Nelumbo nucifera)
Article Snippet: Thereafter, the quality and quantity of RNA were assessed using a NanoPhotometer spectrophotometer (IMPLEN, CA, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). .. These mixed RNA samples were subsequently used in cDNA library construction and Illumina sequencing.

Concentration Assay:

Article Title: Comparative transcript profiling of resistant and susceptible peanut post-harvest seeds in response to aflatoxin production by Aspergillus flavus
Article Snippet: The concentration and integrity of the pooled total RNA was checked using a NanoDrop® 2000 spectrophotometer, a Qubit® Fluorometer 2.0, and an Agilent 2100 bioanalyzer, to confirm that all samples had an RNA integrity number greater than 6.5. .. RNA quality detection, cDNA library construction, and Illumina deep sequencing were performed following the previous method [ , – ].

Article Title: The draft genome assembly of Rhododendron delavayi Franch. var. delavayi
Article Snippet: RNA of each tissue was extracted separately according to the TRIzol protocol (Invitrogen) and then combined in homogenized RNA concentration. .. Both cDNA library construction and Illumina sequencing were carried out by BGI-ShenZhen.

High Throughput Screening Assay:

Article Title: The gut microbiome in atherosclerotic cardiovascular disease
Article Snippet: DNA library construction was performed following the manufacturer’s instruction (Illumina). .. We used the same workflow as described previously to perform cluster generation, template hybridization, isothermal amplification, linearization, blocking and denaturation, and hybridization of the sequencing primers.

Article Title: Impact of a 3-Months Vegetarian Diet on the Gut Microbiota and Immune Repertoire
Article Snippet: DNA library construction was performed following the manufacturer’s instruction (Illumina). .. A similar workflow as described previously ( ) was used to perform cluster generation, template hybridization, isothermal amplification, linearization, blocking and denaturation, and hybridization of the sequencing primers.

Staining:

Article Title: RNA-Seq Uncovers SNPs and Alternative Splicing Events in Asian Lotus (Nelumbo nucifera)
Article Snippet: RNA integrity was evaluated with a 1.0% agarose gel stained with ethidium bromide (EB). .. These mixed RNA samples were subsequently used in cDNA library construction and Illumina sequencing.

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    Illumina Inc dna library construction
    Genome-wide base composition bias curves in <t>Illumina</t> reads from PCR-free human <t>DNA</t> libraries. ( a ) The GC-bias curves from libraries (in duplicate) produced by the immobilized enzyme method (IM-1 and IM-2 in blue), for end repair for 30 min at 20 °C and 3′ A-tailing at 37 °C in contrast to the data from the libraries generated by the soluble enzyme method, with 3′ A-tailing at 65 °C, using enzyme mixture PKT (PKT-1 and PKT-2 in purple). ( b ) The GC-bias data of the immobilized enzyme method compared to the data from the duplicate libraries generated by Illumina TruSeq DNA PCR-free LT Library Preparation Kit (Illumina), Kapa Hyper Prep Kit (Kapa) or NEBNext Ultra II DNA Library Prep Kit for Illumina (Ultra) according to the protocols of the manufacturers. The Illumina protocol carries out end repair for 30 min at 30 °C and 3′ A-tailing for 30 min at 37 °C, followed by incubation at 70 °C for 5 min, and includes a clean-up and size selection step between end repair and 3′ A-tailing. The Kapa Hyper and NEBNext Ultra workflows include an enzyme mixture to perform end repair for 30 min at 20 °C, followed by 3′ A-tailing for 30 min at 65 °C.
    Dna Library Construction, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 98/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc 16s rdna v6
    Enrichment of A. parvulum and altered mitochondrial proteome define the severity of Crohn's disease. ( a ) Interaction network of differentially abundant OTUs; each node represents an OTU and are sized according to their number of interactions; each edge denotes a significant co-exclusion (red) or co-occurence (grey) relationship between OTUs. Nodes are coloured by their number of significant co-exclusions. ( b ) The qPCR quantification of A. parvulum in control and CD microbiota as a function of disease severity. ΔCt was calculated by subtracting average Ct values of universal <t>16S</t> <t>rDNA</t> from average Ct values of A. parvulum specific primers ( n =18 for controls, nine for mild CD and seven for moderate and severe CD). ** P
    16s Rdna V6, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 83/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc rdna
    Contribution of the main phylogenetic groups classified at order level in the controls with no OPE addition (x axis) and in the OPE addition treatments (amendment with 200 ng/L final nominal concentration, y axis) to the total abundance (% total <t>16S</t> <t>rDNA,</t> left panel) and the potential activity (% total rRNA reads, right panel). The values are the average of two replicates. Error bars indicate standard deviation.
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    Genome-wide base composition bias curves in Illumina reads from PCR-free human DNA libraries. ( a ) The GC-bias curves from libraries (in duplicate) produced by the immobilized enzyme method (IM-1 and IM-2 in blue), for end repair for 30 min at 20 °C and 3′ A-tailing at 37 °C in contrast to the data from the libraries generated by the soluble enzyme method, with 3′ A-tailing at 65 °C, using enzyme mixture PKT (PKT-1 and PKT-2 in purple). ( b ) The GC-bias data of the immobilized enzyme method compared to the data from the duplicate libraries generated by Illumina TruSeq DNA PCR-free LT Library Preparation Kit (Illumina), Kapa Hyper Prep Kit (Kapa) or NEBNext Ultra II DNA Library Prep Kit for Illumina (Ultra) according to the protocols of the manufacturers. The Illumina protocol carries out end repair for 30 min at 30 °C and 3′ A-tailing for 30 min at 37 °C, followed by incubation at 70 °C for 5 min, and includes a clean-up and size selection step between end repair and 3′ A-tailing. The Kapa Hyper and NEBNext Ultra workflows include an enzyme mixture to perform end repair for 30 min at 20 °C, followed by 3′ A-tailing for 30 min at 65 °C.

    Journal: Scientific Reports

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

    doi: 10.1038/s41598-018-34079-2

    Figure Lengend Snippet: Genome-wide base composition bias curves in Illumina reads from PCR-free human DNA libraries. ( a ) The GC-bias curves from libraries (in duplicate) produced by the immobilized enzyme method (IM-1 and IM-2 in blue), for end repair for 30 min at 20 °C and 3′ A-tailing at 37 °C in contrast to the data from the libraries generated by the soluble enzyme method, with 3′ A-tailing at 65 °C, using enzyme mixture PKT (PKT-1 and PKT-2 in purple). ( b ) The GC-bias data of the immobilized enzyme method compared to the data from the duplicate libraries generated by Illumina TruSeq DNA PCR-free LT Library Preparation Kit (Illumina), Kapa Hyper Prep Kit (Kapa) or NEBNext Ultra II DNA Library Prep Kit for Illumina (Ultra) according to the protocols of the manufacturers. The Illumina protocol carries out end repair for 30 min at 30 °C and 3′ A-tailing for 30 min at 37 °C, followed by incubation at 70 °C for 5 min, and includes a clean-up and size selection step between end repair and 3′ A-tailing. The Kapa Hyper and NEBNext Ultra workflows include an enzyme mixture to perform end repair for 30 min at 20 °C, followed by 3′ A-tailing for 30 min at 65 °C.

    Article Snippet: To further assess the utility of the immobilized enzymes method on more challenging samples, we have performed Illumina DNA library construction using human FFPE DNA with or without DNA repair as described previously .

    Techniques: Genome Wide, Polymerase Chain Reaction, Gas Chromatography, Produced, Generated, Incubation, Selection

    Effect of end repair and 3′ A-tailing at high temperature on GC-bias in Illumina reads from PCR-free human DNA libraries. ( a ) Comparison of GC-bias curves in duplicate libraries prepared by immobilized enzymes with 3′ A-tailing performed at 37 °C (IM 37 °C -1 and IM 37 °C -2, in blue) or 65 °C (IM 65 °C -1 and IM 65 °C -2, in green) revealed a dramatic effect of 3′ A-tailing at high temperature on sequence coverage of the AT-rich regions from human DNA libraries. ( b ) GC-bias curves were generated from two sets of duplicate libraries produced using the soluble enzyme mixture PKT with (PKT purify-1 and PKT purify-2) or without (PKT-1 and PKT-2) a purification step between end repair and high temperature incubation (with Taq DNA pol added to the samples following purification). Although some bias against AT-rich regions can be attributed to the end repair step, the elevated temperature contributes to the majority of the dropouts in the AT-rich regions. ( c ) Shown are the GC-bias curves from 4 sets of duplicate libraries produced by the method of soluble enzymes. Two sets of the duplicate libraries were purified after end repair with PK mixture and then treated at 37 °C with Klenow Fragment (3′-5′ exo − ) (red, Klenow 37 °C-1 and Klenow 37 °C-2) or Taq DNA pol (blue, Taq 37 °C-1 and Taq 37 °C-2). The other two duplicate sets were prepared using the high temperature treatment protocol either with (green, Taq 65 °C-1 and Taq 65 °C-2) or without (orange, PKT-1 and PKT-2) a purification step between end repair with PKT and treatment with Taq DNA pol at 65 °C for 30 min. ( d ) Comparison of library yield of the three sets described above with or without (PKT on the left) a purification step between end repair and 3′ A-tailing indicates that purification caused substantial loss of library DNA.

    Journal: Scientific Reports

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

    doi: 10.1038/s41598-018-34079-2

    Figure Lengend Snippet: Effect of end repair and 3′ A-tailing at high temperature on GC-bias in Illumina reads from PCR-free human DNA libraries. ( a ) Comparison of GC-bias curves in duplicate libraries prepared by immobilized enzymes with 3′ A-tailing performed at 37 °C (IM 37 °C -1 and IM 37 °C -2, in blue) or 65 °C (IM 65 °C -1 and IM 65 °C -2, in green) revealed a dramatic effect of 3′ A-tailing at high temperature on sequence coverage of the AT-rich regions from human DNA libraries. ( b ) GC-bias curves were generated from two sets of duplicate libraries produced using the soluble enzyme mixture PKT with (PKT purify-1 and PKT purify-2) or without (PKT-1 and PKT-2) a purification step between end repair and high temperature incubation (with Taq DNA pol added to the samples following purification). Although some bias against AT-rich regions can be attributed to the end repair step, the elevated temperature contributes to the majority of the dropouts in the AT-rich regions. ( c ) Shown are the GC-bias curves from 4 sets of duplicate libraries produced by the method of soluble enzymes. Two sets of the duplicate libraries were purified after end repair with PK mixture and then treated at 37 °C with Klenow Fragment (3′-5′ exo − ) (red, Klenow 37 °C-1 and Klenow 37 °C-2) or Taq DNA pol (blue, Taq 37 °C-1 and Taq 37 °C-2). The other two duplicate sets were prepared using the high temperature treatment protocol either with (green, Taq 65 °C-1 and Taq 65 °C-2) or without (orange, PKT-1 and PKT-2) a purification step between end repair with PKT and treatment with Taq DNA pol at 65 °C for 30 min. ( d ) Comparison of library yield of the three sets described above with or without (PKT on the left) a purification step between end repair and 3′ A-tailing indicates that purification caused substantial loss of library DNA.

    Article Snippet: To further assess the utility of the immobilized enzymes method on more challenging samples, we have performed Illumina DNA library construction using human FFPE DNA with or without DNA repair as described previously .

    Techniques: Gas Chromatography, Polymerase Chain Reaction, Sequencing, Generated, Produced, Purification, Incubation

    Enzyme immobilization and comparison of Illumina library preparation protocols. ( a ) A schematic of covalent conjugation of SNAP-tagged enzyme fusion proteins to magnetic beads functionalized with O 6 -benzylguanine (BG) moieties that specifically react with active site cysteine residues of SNAP-tag proteins, forming a stable covalent thioether bond 15 , 16 . ( b ) Workflow for library construction using immobilized enzymes for Illumina sequencing. A typical streamlined protocol for Illumina library construction is modified by employing immobilized enzymes to catalyze end repair and 3′ A-tailing. This method utilizes SNAP-tagged T4 DNA pol and PNK covalently conjugated to BG-functionalized magnetic beads to carry out end repair of fragmented DNA at 20°C (or 37 °C) for 30 min. The enzymes are removed by magnetic separation from the DNA pool, which is subjected to 3′ A-tailing at 37 °C for 30 min using immobilized Taq DNA pol. ( c ) Streamlined protocol for Illumina amplification-free library preparation using soluble enzymes. Typically, end repair and 3′ A-tailing of fragmented DNA are catalyzed by an enzyme mixture at 20 °C for 30 min, followed by heat treatment at 65 °C for 30 min. ( d ) The workflow of Illumina TruSeq DNA PCR-free LT Library Prep Kit with a purification step. End repair is performed for 30 min at 30 °C, followed by a bead-based step for clean up and size selection. 3′ A-tailing is carried out for 30 min at 37 °C with a subsequent treatment for 5 min at 70 °C. Each library was ligated to preannealed full-length paired-end Illumina adaptors, size-selected and analyzed, and sequenced on an Illumina sequencing platform.

    Journal: Scientific Reports

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

    doi: 10.1038/s41598-018-34079-2

    Figure Lengend Snippet: Enzyme immobilization and comparison of Illumina library preparation protocols. ( a ) A schematic of covalent conjugation of SNAP-tagged enzyme fusion proteins to magnetic beads functionalized with O 6 -benzylguanine (BG) moieties that specifically react with active site cysteine residues of SNAP-tag proteins, forming a stable covalent thioether bond 15 , 16 . ( b ) Workflow for library construction using immobilized enzymes for Illumina sequencing. A typical streamlined protocol for Illumina library construction is modified by employing immobilized enzymes to catalyze end repair and 3′ A-tailing. This method utilizes SNAP-tagged T4 DNA pol and PNK covalently conjugated to BG-functionalized magnetic beads to carry out end repair of fragmented DNA at 20°C (or 37 °C) for 30 min. The enzymes are removed by magnetic separation from the DNA pool, which is subjected to 3′ A-tailing at 37 °C for 30 min using immobilized Taq DNA pol. ( c ) Streamlined protocol for Illumina amplification-free library preparation using soluble enzymes. Typically, end repair and 3′ A-tailing of fragmented DNA are catalyzed by an enzyme mixture at 20 °C for 30 min, followed by heat treatment at 65 °C for 30 min. ( d ) The workflow of Illumina TruSeq DNA PCR-free LT Library Prep Kit with a purification step. End repair is performed for 30 min at 30 °C, followed by a bead-based step for clean up and size selection. 3′ A-tailing is carried out for 30 min at 37 °C with a subsequent treatment for 5 min at 70 °C. Each library was ligated to preannealed full-length paired-end Illumina adaptors, size-selected and analyzed, and sequenced on an Illumina sequencing platform.

    Article Snippet: To further assess the utility of the immobilized enzymes method on more challenging samples, we have performed Illumina DNA library construction using human FFPE DNA with or without DNA repair as described previously .

    Techniques: Conjugation Assay, Magnetic Beads, Sequencing, Modification, Amplification, Polymerase Chain Reaction, Purification, Selection

    Enrichment of A. parvulum and altered mitochondrial proteome define the severity of Crohn's disease. ( a ) Interaction network of differentially abundant OTUs; each node represents an OTU and are sized according to their number of interactions; each edge denotes a significant co-exclusion (red) or co-occurence (grey) relationship between OTUs. Nodes are coloured by their number of significant co-exclusions. ( b ) The qPCR quantification of A. parvulum in control and CD microbiota as a function of disease severity. ΔCt was calculated by subtracting average Ct values of universal 16S rDNA from average Ct values of A. parvulum specific primers ( n =18 for controls, nine for mild CD and seven for moderate and severe CD). ** P

    Journal: Nature Communications

    Article Title: Altered intestinal microbiota–host mitochondria crosstalk in new onset Crohn's disease

    doi: 10.1038/ncomms13419

    Figure Lengend Snippet: Enrichment of A. parvulum and altered mitochondrial proteome define the severity of Crohn's disease. ( a ) Interaction network of differentially abundant OTUs; each node represents an OTU and are sized according to their number of interactions; each edge denotes a significant co-exclusion (red) or co-occurence (grey) relationship between OTUs. Nodes are coloured by their number of significant co-exclusions. ( b ) The qPCR quantification of A. parvulum in control and CD microbiota as a function of disease severity. ΔCt was calculated by subtracting average Ct values of universal 16S rDNA from average Ct values of A. parvulum specific primers ( n =18 for controls, nine for mild CD and seven for moderate and severe CD). ** P

    Article Snippet: For the V6 reactions, the PCR cycling conditions were identical to the first PCR reaction described in the 16S rDNA-V6 Illumina library construction ‘Methods' section.

    Techniques: Real-time Polymerase Chain Reaction

    Contribution of the main phylogenetic groups classified at order level in the controls with no OPE addition (x axis) and in the OPE addition treatments (amendment with 200 ng/L final nominal concentration, y axis) to the total abundance (% total 16S rDNA, left panel) and the potential activity (% total rRNA reads, right panel). The values are the average of two replicates. Error bars indicate standard deviation.

    Journal: Scientific Reports

    Article Title: Microbial consumption of organophosphate esters in seawater under phosphorus limited conditions

    doi: 10.1038/s41598-018-36635-2

    Figure Lengend Snippet: Contribution of the main phylogenetic groups classified at order level in the controls with no OPE addition (x axis) and in the OPE addition treatments (amendment with 200 ng/L final nominal concentration, y axis) to the total abundance (% total 16S rDNA, left panel) and the potential activity (% total rRNA reads, right panel). The values are the average of two replicates. Error bars indicate standard deviation.

    Article Snippet: Samples for 16S rRNA and rDNA Illumina MiSeq library construction and for determination of OPEs concentrations were collected 0.5 and 24 hours after addition of seawater.

    Techniques: Concentration Assay, Activity Assay, Standard Deviation

    Contribution of the main phylogenetic groups classified at order level in the controls with no OPE addition (x axis) and in the OPE addition treatments (amendment with 200 ng/L final nominal concentration, y axis) to the total abundance (% total 16S rDNA, left panel) and the potential activity (% total rRNA reads, right panel). The values are the average of two replicates. Error bars indicate standard deviation.

    Journal: Scientific Reports

    Article Title: Microbial consumption of organophosphate esters in seawater under phosphorus limited conditions

    doi: 10.1038/s41598-018-36635-2

    Figure Lengend Snippet: Contribution of the main phylogenetic groups classified at order level in the controls with no OPE addition (x axis) and in the OPE addition treatments (amendment with 200 ng/L final nominal concentration, y axis) to the total abundance (% total 16S rDNA, left panel) and the potential activity (% total rRNA reads, right panel). The values are the average of two replicates. Error bars indicate standard deviation.

    Article Snippet: Samples for 16S rRNA and rDNA Illumina MiSeq library construction and for determination of OPEs concentrations were collected 0.5 and 24 hours after addition of seawater.

    Techniques: Concentration Assay, Activity Assay, Standard Deviation