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Illumina Inc genomic dna primer
Comparison of input <t>DNA</t> signal tracks among all four barcoded adapters relative to standard <t>Illumina</t> adapters . An input sample was split in five aliquots. Four were barcoded differentially (top four lanes) and one had non-barcoded, Illumina adapters (fifth lane, labeled 'None'). Barcoded inputs were scored against non-barcoded input. IGB signal tracks of yeast chromosome 16 are shown for each sample, with ORF locations on the x-axis. ORFs are depicted in purple. On the y-axis, a normalized scale represents the number of read counts at a particular location. Each scale is normalized according to the number of mapped reads (Table 10 ). A box in the left panel depicts the enlarged section shown in the right panel for positions between 828,000 and 833,000 to demonstrate the overlap among all signal tracks.
Genomic Dna Primer, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing"

Article Title: Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing

Journal: BMC Genomics

doi: 10.1186/1471-2164-10-37

Comparison of input DNA signal tracks among all four barcoded adapters relative to standard Illumina adapters . An input sample was split in five aliquots. Four were barcoded differentially (top four lanes) and one had non-barcoded, Illumina adapters (fifth lane, labeled 'None'). Barcoded inputs were scored against non-barcoded input. IGB signal tracks of yeast chromosome 16 are shown for each sample, with ORF locations on the x-axis. ORFs are depicted in purple. On the y-axis, a normalized scale represents the number of read counts at a particular location. Each scale is normalized according to the number of mapped reads (Table 10 ). A box in the left panel depicts the enlarged section shown in the right panel for positions between 828,000 and 833,000 to demonstrate the overlap among all signal tracks.
Figure Legend Snippet: Comparison of input DNA signal tracks among all four barcoded adapters relative to standard Illumina adapters . An input sample was split in five aliquots. Four were barcoded differentially (top four lanes) and one had non-barcoded, Illumina adapters (fifth lane, labeled 'None'). Barcoded inputs were scored against non-barcoded input. IGB signal tracks of yeast chromosome 16 are shown for each sample, with ORF locations on the x-axis. ORFs are depicted in purple. On the y-axis, a normalized scale represents the number of read counts at a particular location. Each scale is normalized according to the number of mapped reads (Table 10 ). A box in the left panel depicts the enlarged section shown in the right panel for positions between 828,000 and 833,000 to demonstrate the overlap among all signal tracks.

Techniques Used: Labeling

Barcoded adapters perform similarly to standard Illumina adapters and do not crossover to other samples in the same lane . (a) RNA PolII binding profiles from different biological replicates with the same barcode (PolII_Rep1, dark blue; PolII_Rep3, red), with different barcodes (PolII_Rep2, orange) or without barcode (PolII_Rep4, green) strongly overlap. See also Table 3 . Input DNA serves as a reference (light blue). IGB signal tracks of chromosome 5 between 130,000 and 320,000 are shown for each library. A box in the left panel depicts the enlarged section shown in the right panel between positions 298,000 and 309,000 to illustrate the overlap among all PolII signal tracks. (b) Binding profiles from four different libraries pooled and sequenced in the same flowcell lane show very little resemblance. Shown here are the binding profiles for Cse4_Rep2 (dark blue), Ste12_Rep2 (red), PolII_Rep2 (green) and Input_ACGT (light blue). IGB signal tracks of chromosome 12 between 80,000 and 210,000 are shown for each sample. For (a) and (b), axis and scale normalizations are similar to Figure 2 . (c) Left: Rank-rank comparison of target lists between all pairwise barcoded replicates for Cse4, PolII and Ste12. The horizontal axis shows the fraction of the two lists being compared and the vertical axis shows the fraction of those targets that agree between a given pair of target lists. All comparisons show strong agreement, although the rank lists for Cse4 differ more than PolII or Ste12 for the second half of their length. Right: Rank-rank comparison between barcoded replicates from the same factors (averaged over all pairwise comparisons) compared to rank-rank comparisons for barcoded replicates between different factors: PolII_Rep1 (ACGT) vs. Ste12_Rep1 (TGCT) and Cse4_Rep2 (CATT) vs. Ste12_Rep2 (GTAT).
Figure Legend Snippet: Barcoded adapters perform similarly to standard Illumina adapters and do not crossover to other samples in the same lane . (a) RNA PolII binding profiles from different biological replicates with the same barcode (PolII_Rep1, dark blue; PolII_Rep3, red), with different barcodes (PolII_Rep2, orange) or without barcode (PolII_Rep4, green) strongly overlap. See also Table 3 . Input DNA serves as a reference (light blue). IGB signal tracks of chromosome 5 between 130,000 and 320,000 are shown for each library. A box in the left panel depicts the enlarged section shown in the right panel between positions 298,000 and 309,000 to illustrate the overlap among all PolII signal tracks. (b) Binding profiles from four different libraries pooled and sequenced in the same flowcell lane show very little resemblance. Shown here are the binding profiles for Cse4_Rep2 (dark blue), Ste12_Rep2 (red), PolII_Rep2 (green) and Input_ACGT (light blue). IGB signal tracks of chromosome 12 between 80,000 and 210,000 are shown for each sample. For (a) and (b), axis and scale normalizations are similar to Figure 2 . (c) Left: Rank-rank comparison of target lists between all pairwise barcoded replicates for Cse4, PolII and Ste12. The horizontal axis shows the fraction of the two lists being compared and the vertical axis shows the fraction of those targets that agree between a given pair of target lists. All comparisons show strong agreement, although the rank lists for Cse4 differ more than PolII or Ste12 for the second half of their length. Right: Rank-rank comparison between barcoded replicates from the same factors (averaged over all pairwise comparisons) compared to rank-rank comparisons for barcoded replicates between different factors: PolII_Rep1 (ACGT) vs. Ste12_Rep1 (TGCT) and Cse4_Rep2 (CATT) vs. Ste12_Rep2 (GTAT).

Techniques Used: Binding Assay

Scheme for yeast barcoded ChIP-Seq . (a) Barcoded ChIP-Seq workflow. Ovals depict yeast cells and squares depict proteins. An aliquot of sheared cell lysate is not immunoprecipitated but is otherwise processed normally (green). This DNA, termed input DNA, is a reference sample for ChIP-Seq. Illumina DNA libraries are generated from both ChIP and input DNA samples. In multiplex ChIP-Seq, a barcoded adapter is ligated to an individual DNA sample. The barcode has 3 unique bases followed by a 'T' to anneal with the end-repaired DNA. Four libraries are then pooled together and applied to a single flowcell lane. After sequencing on the Genome Analyzer, reads are separated according to the first four bases and aligned to the yeast genome. Reads are stacked to generate a signal profile and scored against a pool of input DNA to determine significant transcription factor binding sites. (b) The barcode (orange) is located between Illumina adapter sequences (purple) and ChIP or input DNA inserts (black). The sequencing primer (pink) anneals to the adapter sequences and short reads start with the four bases of the barcode (orange) followed by DNA inserts (black). For the sequencing primer and Illumina adapter, oligonucleotide sequences were given by the manufacturer © 2006 Illumina, Inc. All rights reserved.
Figure Legend Snippet: Scheme for yeast barcoded ChIP-Seq . (a) Barcoded ChIP-Seq workflow. Ovals depict yeast cells and squares depict proteins. An aliquot of sheared cell lysate is not immunoprecipitated but is otherwise processed normally (green). This DNA, termed input DNA, is a reference sample for ChIP-Seq. Illumina DNA libraries are generated from both ChIP and input DNA samples. In multiplex ChIP-Seq, a barcoded adapter is ligated to an individual DNA sample. The barcode has 3 unique bases followed by a 'T' to anneal with the end-repaired DNA. Four libraries are then pooled together and applied to a single flowcell lane. After sequencing on the Genome Analyzer, reads are separated according to the first four bases and aligned to the yeast genome. Reads are stacked to generate a signal profile and scored against a pool of input DNA to determine significant transcription factor binding sites. (b) The barcode (orange) is located between Illumina adapter sequences (purple) and ChIP or input DNA inserts (black). The sequencing primer (pink) anneals to the adapter sequences and short reads start with the four bases of the barcode (orange) followed by DNA inserts (black). For the sequencing primer and Illumina adapter, oligonucleotide sequences were given by the manufacturer © 2006 Illumina, Inc. All rights reserved.

Techniques Used: Chromatin Immunoprecipitation, Immunoprecipitation, Generated, Multiplex Assay, Sequencing, Binding Assay

2) Product Images from "Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing"

Article Title: Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing

Journal: BMC Genomics

doi: 10.1186/1471-2164-10-37

Comparison of input DNA signal tracks among all four barcoded adapters relative to standard Illumina adapters . An input sample was split in five aliquots. Four were barcoded differentially (top four lanes) and one had non-barcoded, Illumina adapters (fifth lane, labeled 'None'). Barcoded inputs were scored against non-barcoded input. IGB signal tracks of yeast chromosome 16 are shown for each sample, with ORF locations on the x-axis. ORFs are depicted in purple. On the y-axis, a normalized scale represents the number of read counts at a particular location. Each scale is normalized according to the number of mapped reads (Table 10 ). A box in the left panel depicts the enlarged section shown in the right panel for positions between 828,000 and 833,000 to demonstrate the overlap among all signal tracks.
Figure Legend Snippet: Comparison of input DNA signal tracks among all four barcoded adapters relative to standard Illumina adapters . An input sample was split in five aliquots. Four were barcoded differentially (top four lanes) and one had non-barcoded, Illumina adapters (fifth lane, labeled 'None'). Barcoded inputs were scored against non-barcoded input. IGB signal tracks of yeast chromosome 16 are shown for each sample, with ORF locations on the x-axis. ORFs are depicted in purple. On the y-axis, a normalized scale represents the number of read counts at a particular location. Each scale is normalized according to the number of mapped reads (Table 10 ). A box in the left panel depicts the enlarged section shown in the right panel for positions between 828,000 and 833,000 to demonstrate the overlap among all signal tracks.

Techniques Used: Labeling

Barcoded adapters perform similarly to standard Illumina adapters and do not crossover to other samples in the same lane . (a) RNA PolII binding profiles from different biological replicates with the same barcode (PolII_Rep1, dark blue; PolII_Rep3, red), with different barcodes (PolII_Rep2, orange) or without barcode (PolII_Rep4, green) strongly overlap. See also Table 3 . Input DNA serves as a reference (light blue). IGB signal tracks of chromosome 5 between 130,000 and 320,000 are shown for each library. A box in the left panel depicts the enlarged section shown in the right panel between positions 298,000 and 309,000 to illustrate the overlap among all PolII signal tracks. (b) Binding profiles from four different libraries pooled and sequenced in the same flowcell lane show very little resemblance. Shown here are the binding profiles for Cse4_Rep2 (dark blue), Ste12_Rep2 (red), PolII_Rep2 (green) and Input_ACGT (light blue). IGB signal tracks of chromosome 12 between 80,000 and 210,000 are shown for each sample. For (a) and (b), axis and scale normalizations are similar to Figure 2 . (c) Left: Rank-rank comparison of target lists between all pairwise barcoded replicates for Cse4, PolII and Ste12. The horizontal axis shows the fraction of the two lists being compared and the vertical axis shows the fraction of those targets that agree between a given pair of target lists. All comparisons show strong agreement, although the rank lists for Cse4 differ more than PolII or Ste12 for the second half of their length. Right: Rank-rank comparison between barcoded replicates from the same factors (averaged over all pairwise comparisons) compared to rank-rank comparisons for barcoded replicates between different factors: PolII_Rep1 (ACGT) vs. Ste12_Rep1 (TGCT) and Cse4_Rep2 (CATT) vs. Ste12_Rep2 (GTAT).
Figure Legend Snippet: Barcoded adapters perform similarly to standard Illumina adapters and do not crossover to other samples in the same lane . (a) RNA PolII binding profiles from different biological replicates with the same barcode (PolII_Rep1, dark blue; PolII_Rep3, red), with different barcodes (PolII_Rep2, orange) or without barcode (PolII_Rep4, green) strongly overlap. See also Table 3 . Input DNA serves as a reference (light blue). IGB signal tracks of chromosome 5 between 130,000 and 320,000 are shown for each library. A box in the left panel depicts the enlarged section shown in the right panel between positions 298,000 and 309,000 to illustrate the overlap among all PolII signal tracks. (b) Binding profiles from four different libraries pooled and sequenced in the same flowcell lane show very little resemblance. Shown here are the binding profiles for Cse4_Rep2 (dark blue), Ste12_Rep2 (red), PolII_Rep2 (green) and Input_ACGT (light blue). IGB signal tracks of chromosome 12 between 80,000 and 210,000 are shown for each sample. For (a) and (b), axis and scale normalizations are similar to Figure 2 . (c) Left: Rank-rank comparison of target lists between all pairwise barcoded replicates for Cse4, PolII and Ste12. The horizontal axis shows the fraction of the two lists being compared and the vertical axis shows the fraction of those targets that agree between a given pair of target lists. All comparisons show strong agreement, although the rank lists for Cse4 differ more than PolII or Ste12 for the second half of their length. Right: Rank-rank comparison between barcoded replicates from the same factors (averaged over all pairwise comparisons) compared to rank-rank comparisons for barcoded replicates between different factors: PolII_Rep1 (ACGT) vs. Ste12_Rep1 (TGCT) and Cse4_Rep2 (CATT) vs. Ste12_Rep2 (GTAT).

Techniques Used: Binding Assay

Scheme for yeast barcoded ChIP-Seq . (a) Barcoded ChIP-Seq workflow. Ovals depict yeast cells and squares depict proteins. An aliquot of sheared cell lysate is not immunoprecipitated but is otherwise processed normally (green). This DNA, termed input DNA, is a reference sample for ChIP-Seq. Illumina DNA libraries are generated from both ChIP and input DNA samples. In multiplex ChIP-Seq, a barcoded adapter is ligated to an individual DNA sample. The barcode has 3 unique bases followed by a 'T' to anneal with the end-repaired DNA. Four libraries are then pooled together and applied to a single flowcell lane. After sequencing on the Genome Analyzer, reads are separated according to the first four bases and aligned to the yeast genome. Reads are stacked to generate a signal profile and scored against a pool of input DNA to determine significant transcription factor binding sites. (b) The barcode (orange) is located between Illumina adapter sequences (purple) and ChIP or input DNA inserts (black). The sequencing primer (pink) anneals to the adapter sequences and short reads start with the four bases of the barcode (orange) followed by DNA inserts (black). For the sequencing primer and Illumina adapter, oligonucleotide sequences were given by the manufacturer © 2006 Illumina, Inc. All rights reserved.
Figure Legend Snippet: Scheme for yeast barcoded ChIP-Seq . (a) Barcoded ChIP-Seq workflow. Ovals depict yeast cells and squares depict proteins. An aliquot of sheared cell lysate is not immunoprecipitated but is otherwise processed normally (green). This DNA, termed input DNA, is a reference sample for ChIP-Seq. Illumina DNA libraries are generated from both ChIP and input DNA samples. In multiplex ChIP-Seq, a barcoded adapter is ligated to an individual DNA sample. The barcode has 3 unique bases followed by a 'T' to anneal with the end-repaired DNA. Four libraries are then pooled together and applied to a single flowcell lane. After sequencing on the Genome Analyzer, reads are separated according to the first four bases and aligned to the yeast genome. Reads are stacked to generate a signal profile and scored against a pool of input DNA to determine significant transcription factor binding sites. (b) The barcode (orange) is located between Illumina adapter sequences (purple) and ChIP or input DNA inserts (black). The sequencing primer (pink) anneals to the adapter sequences and short reads start with the four bases of the barcode (orange) followed by DNA inserts (black). For the sequencing primer and Illumina adapter, oligonucleotide sequences were given by the manufacturer © 2006 Illumina, Inc. All rights reserved.

Techniques Used: Chromatin Immunoprecipitation, Immunoprecipitation, Generated, Multiplex Assay, Sequencing, Binding Assay

Related Articles

Flow Cytometry:

Article Title: H3K9 and H3K14 acetylation co-occur at many gene regulatory elements, while H3K14ac marks a subset of inactive inducible promoters in mouse embryonic stem cells
Article Snippet: To amplify the library, PCR was performed using Illumina genomic DNA primer "1.1" and Illumina genomic DNA primer "2.1" with 15 cycles (Input DNA) or 17 cycles (ChIP DNA) of amplification. .. A final size selection was performed using a 2% agarose E-Gel to obtain a library with a median length of ~230 bp which is within the recommended size range for cluster generation on Illumina's flow cell.

Amplification:

Article Title: Whole transcriptome analysis with sequencing: methods, challenges and potential solutions
Article Snippet: The amplified products were fragmented and size-selected for 100–300 bp, which were then end-repaired, dA-tailed and ligated with the Illumina sequencing adaptors ( ). .. PCR was performed again using Illumina’s genomic DNA primer set for cluster generation and sequencing on the Illumina Cluster Station and Illumina Genome Analyzer I.

Article Title: H3K9 and H3K14 acetylation co-occur at many gene regulatory elements, while H3K14ac marks a subset of inactive inducible promoters in mouse embryonic stem cells
Article Snippet: .. To amplify the library, PCR was performed using Illumina genomic DNA primer "1.1" and Illumina genomic DNA primer "2.1" with 15 cycles (Input DNA) or 17 cycles (ChIP DNA) of amplification. .. A final size selection was performed using a 2% agarose E-Gel to obtain a library with a median length of ~230 bp which is within the recommended size range for cluster generation on Illumina's flow cell.


RNA Sequencing Assay:

Article Title: Whole transcriptome analysis with sequencing: methods, challenges and potential solutions
Article Snippet: Paragraph title: RNA-seq ... PCR was performed again using Illumina’s genomic DNA primer set for cluster generation and sequencing on the Illumina Cluster Station and Illumina Genome Analyzer I.

Ligation:


Article Title: Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing
Article Snippet: We found that size-selection and purification of the ligation products by agarose gel before amplification of the library by PCR resulted in better libraries and decreased the incidence and intensity of an adapter-adapter band at ~120 bp. .. To amplify the library, PCR was performed using Illumina genomic DNA primer "1.1" and Illumina genomic DNA primer "2.1" as described [ , ] with 15 cycles (Input DNA) or 17 cycles (ChIP DNA) of amplification.

Polymerase Chain Reaction:

Article Title: Whole transcriptome analysis with sequencing: methods, challenges and potential solutions
Article Snippet: .. PCR was performed again using Illumina’s genomic DNA primer set for cluster generation and sequencing on the Illumina Cluster Station and Illumina Genome Analyzer I. .. The whole transcriptome shotgun sequencing technique, now known as the RNA-Seq method has dramatically shaped the landscape of whole transcriptome profiling [ – ].

Article Title: H3K9 and H3K14 acetylation co-occur at many gene regulatory elements, while H3K14ac marks a subset of inactive inducible promoters in mouse embryonic stem cells
Article Snippet: .. To amplify the library, PCR was performed using Illumina genomic DNA primer "1.1" and Illumina genomic DNA primer "2.1" with 15 cycles (Input DNA) or 17 cycles (ChIP DNA) of amplification. .. A final size selection was performed using a 2% agarose E-Gel to obtain a library with a median length of ~230 bp which is within the recommended size range for cluster generation on Illumina's flow cell.

Article Title: Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing
Article Snippet: .. To amplify the library, PCR was performed using Illumina genomic DNA primer "1.1" and Illumina genomic DNA primer "2.1" as described [ , ] with 15 cycles (Input DNA) or 17 cycles (ChIP DNA) of amplification. .. A final size selection was performed using a 2% agarose E-Gel to obtain a library with a median length of ~230 bp which is within the recommended size range for cluster generation on Illumina's flowcell.

Construct:

Article Title: Whole transcriptome analysis with sequencing: methods, challenges and potential solutions
Article Snippet: Morin and colleagues [ ] constructed four libraries for whole transcriptome shotgun sequencing using randomly primed cDNA and massively parallel short-read sequencing on an Illumina Genome Analyzer I. .. PCR was performed again using Illumina’s genomic DNA primer set for cluster generation and sequencing on the Illumina Cluster Station and Illumina Genome Analyzer I.

Purification:

Article Title: H3K9 and H3K14 acetylation co-occur at many gene regulatory elements, while H3K14ac marks a subset of inactive inducible promoters in mouse embryonic stem cells
Article Snippet: After 15 min, samples were purified with the MinElute PCR purification kit (Qiagen). .. To amplify the library, PCR was performed using Illumina genomic DNA primer "1.1" and Illumina genomic DNA primer "2.1" with 15 cycles (Input DNA) or 17 cycles (ChIP DNA) of amplification.

Article Title: Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing
Article Snippet: We found that size-selection and purification of the ligation products by agarose gel before amplification of the library by PCR resulted in better libraries and decreased the incidence and intensity of an adapter-adapter band at ~120 bp. .. To amplify the library, PCR was performed using Illumina genomic DNA primer "1.1" and Illumina genomic DNA primer "2.1" as described [ , ] with 15 cycles (Input DNA) or 17 cycles (ChIP DNA) of amplification.

Sequencing:

Article Title: Whole transcriptome analysis with sequencing: methods, challenges and potential solutions
Article Snippet: .. PCR was performed again using Illumina’s genomic DNA primer set for cluster generation and sequencing on the Illumina Cluster Station and Illumina Genome Analyzer I. .. The whole transcriptome shotgun sequencing technique, now known as the RNA-Seq method has dramatically shaped the landscape of whole transcriptome profiling [ – ].

Article Title: H3K9 and H3K14 acetylation co-occur at many gene regulatory elements, while H3K14ac marks a subset of inactive inducible promoters in mouse embryonic stem cells
Article Snippet: Paragraph title: Library preparation and sequencing ... To amplify the library, PCR was performed using Illumina genomic DNA primer "1.1" and Illumina genomic DNA primer "2.1" with 15 cycles (Input DNA) or 17 cycles (ChIP DNA) of amplification.

Selection:

Article Title: H3K9 and H3K14 acetylation co-occur at many gene regulatory elements, while H3K14ac marks a subset of inactive inducible promoters in mouse embryonic stem cells
Article Snippet: To amplify the library, PCR was performed using Illumina genomic DNA primer "1.1" and Illumina genomic DNA primer "2.1" with 15 cycles (Input DNA) or 17 cycles (ChIP DNA) of amplification. .. A final size selection was performed using a 2% agarose E-Gel to obtain a library with a median length of ~230 bp which is within the recommended size range for cluster generation on Illumina's flow cell.

Article Title: Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing
Article Snippet: To amplify the library, PCR was performed using Illumina genomic DNA primer "1.1" and Illumina genomic DNA primer "2.1" as described [ , ] with 15 cycles (Input DNA) or 17 cycles (ChIP DNA) of amplification. .. A final size selection was performed using a 2% agarose E-Gel to obtain a library with a median length of ~230 bp which is within the recommended size range for cluster generation on Illumina's flowcell.

Spectrophotometry:

Article Title: H3K9 and H3K14 acetylation co-occur at many gene regulatory elements, while H3K14ac marks a subset of inactive inducible promoters in mouse embryonic stem cells
Article Snippet: To amplify the library, PCR was performed using Illumina genomic DNA primer "1.1" and Illumina genomic DNA primer "2.1" with 15 cycles (Input DNA) or 17 cycles (ChIP DNA) of amplification. .. Finally, DNA concentrations and purities (A260/280 nm ratios) were measured on a Nanodrop spectrophotometer.

Gel Extraction:

Article Title: H3K9 and H3K14 acetylation co-occur at many gene regulatory elements, while H3K14ac marks a subset of inactive inducible promoters in mouse embryonic stem cells
Article Snippet: DNA fragments ranging from 150 base pairs to 500 base pairs were extracted and recovered in 28 μl EB with a QIAquick gel extraction kit (Qiagen). .. To amplify the library, PCR was performed using Illumina genomic DNA primer "1.1" and Illumina genomic DNA primer "2.1" with 15 cycles (Input DNA) or 17 cycles (ChIP DNA) of amplification.

Article Title: Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing
Article Snippet: DNA fragments ranging from 150 base pairs to 500 base pairs were extracted and recovered in 28 μL EB with a QIAquick gel extraction kit (Qiagen). .. To amplify the library, PCR was performed using Illumina genomic DNA primer "1.1" and Illumina genomic DNA primer "2.1" as described [ , ] with 15 cycles (Input DNA) or 17 cycles (ChIP DNA) of amplification.

Modification:

Article Title: Whole transcriptome analysis with sequencing: methods, challenges and potential solutions
Article Snippet: Basically, both modified oligo(dT) and template-switching primers were used to produce full-length single-stranded cDNAs containing the complete 5′ end of the mRNA and universal priming sequences for end-to-end PCR amplification. .. PCR was performed again using Illumina’s genomic DNA primer set for cluster generation and sequencing on the Illumina Cluster Station and Illumina Genome Analyzer I.

Agarose Gel Electrophoresis:

Article Title: Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing
Article Snippet: We found that size-selection and purification of the ligation products by agarose gel before amplification of the library by PCR resulted in better libraries and decreased the incidence and intensity of an adapter-adapter band at ~120 bp. .. To amplify the library, PCR was performed using Illumina genomic DNA primer "1.1" and Illumina genomic DNA primer "2.1" as described [ , ] with 15 cycles (Input DNA) or 17 cycles (ChIP DNA) of amplification.

Shotgun Sequencing:

Article Title: Whole transcriptome analysis with sequencing: methods, challenges and potential solutions
Article Snippet: Morin and colleagues [ ] constructed four libraries for whole transcriptome shotgun sequencing using randomly primed cDNA and massively parallel short-read sequencing on an Illumina Genome Analyzer I. .. PCR was performed again using Illumina’s genomic DNA primer set for cluster generation and sequencing on the Illumina Cluster Station and Illumina Genome Analyzer I.

Chromatin Immunoprecipitation:

Article Title: H3K9 and H3K14 acetylation co-occur at many gene regulatory elements, while H3K14ac marks a subset of inactive inducible promoters in mouse embryonic stem cells
Article Snippet: .. To amplify the library, PCR was performed using Illumina genomic DNA primer "1.1" and Illumina genomic DNA primer "2.1" with 15 cycles (Input DNA) or 17 cycles (ChIP DNA) of amplification. .. A final size selection was performed using a 2% agarose E-Gel to obtain a library with a median length of ~230 bp which is within the recommended size range for cluster generation on Illumina's flow cell.

Article Title: Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing
Article Snippet: .. To amplify the library, PCR was performed using Illumina genomic DNA primer "1.1" and Illumina genomic DNA primer "2.1" as described [ , ] with 15 cycles (Input DNA) or 17 cycles (ChIP DNA) of amplification. .. A final size selection was performed using a 2% agarose E-Gel to obtain a library with a median length of ~230 bp which is within the recommended size range for cluster generation on Illumina's flowcell.

Gel Purification:

Article Title: H3K9 and H3K14 acetylation co-occur at many gene regulatory elements, while H3K14ac marks a subset of inactive inducible promoters in mouse embryonic stem cells
Article Snippet: Adapters in excess were eliminated by using gel purification on a 2% agarose E-Gel (Invitrogen) for 20 min, together with Track-It 50 bp DNA ladder (Invitrogen). .. To amplify the library, PCR was performed using Illumina genomic DNA primer "1.1" and Illumina genomic DNA primer "2.1" with 15 cycles (Input DNA) or 17 cycles (ChIP DNA) of amplification.

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    Illumina Inc genomic dna primer
    Comparison of input <t>DNA</t> signal tracks among all four barcoded adapters relative to standard <t>Illumina</t> adapters . An input sample was split in five aliquots. Four were barcoded differentially (top four lanes) and one had non-barcoded, Illumina adapters (fifth lane, labeled 'None'). Barcoded inputs were scored against non-barcoded input. IGB signal tracks of yeast chromosome 16 are shown for each sample, with ORF locations on the x-axis. ORFs are depicted in purple. On the y-axis, a normalized scale represents the number of read counts at a particular location. Each scale is normalized according to the number of mapped reads (Table 10 ). A box in the left panel depicts the enlarged section shown in the right panel for positions between 828,000 and 833,000 to demonstrate the overlap among all signal tracks.
    Genomic Dna Primer, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/genomic dna primer/product/Illumina Inc
    Average 88 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    genomic dna primer - by Bioz Stars, 2020-04
    88/100 stars
      Buy from Supplier

    87
    Illumina Inc genomic dna sequencing primer
    Schematic description of the high-throughput <t>SELEX</t> process. ( A ) Protein expression. ( Top ) Proteins are expressed as fusion proteins with SBP-tagged Gaussia -luciferase. ( Bottom ) The GATEWAY recombination cloning system is used to transfer <t>DNA</t> sequences
    Genomic Dna Sequencing Primer, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 87/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/genomic dna sequencing primer/product/Illumina Inc
    Average 87 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    genomic dna sequencing primer - by Bioz Stars, 2020-04
    87/100 stars
      Buy from Supplier

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    Comparison of input DNA signal tracks among all four barcoded adapters relative to standard Illumina adapters . An input sample was split in five aliquots. Four were barcoded differentially (top four lanes) and one had non-barcoded, Illumina adapters (fifth lane, labeled 'None'). Barcoded inputs were scored against non-barcoded input. IGB signal tracks of yeast chromosome 16 are shown for each sample, with ORF locations on the x-axis. ORFs are depicted in purple. On the y-axis, a normalized scale represents the number of read counts at a particular location. Each scale is normalized according to the number of mapped reads (Table 10 ). A box in the left panel depicts the enlarged section shown in the right panel for positions between 828,000 and 833,000 to demonstrate the overlap among all signal tracks.

    Journal: BMC Genomics

    Article Title: Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing

    doi: 10.1186/1471-2164-10-37

    Figure Lengend Snippet: Comparison of input DNA signal tracks among all four barcoded adapters relative to standard Illumina adapters . An input sample was split in five aliquots. Four were barcoded differentially (top four lanes) and one had non-barcoded, Illumina adapters (fifth lane, labeled 'None'). Barcoded inputs were scored against non-barcoded input. IGB signal tracks of yeast chromosome 16 are shown for each sample, with ORF locations on the x-axis. ORFs are depicted in purple. On the y-axis, a normalized scale represents the number of read counts at a particular location. Each scale is normalized according to the number of mapped reads (Table 10 ). A box in the left panel depicts the enlarged section shown in the right panel for positions between 828,000 and 833,000 to demonstrate the overlap among all signal tracks.

    Article Snippet: To amplify the library, PCR was performed using Illumina genomic DNA primer "1.1" and Illumina genomic DNA primer "2.1" as described [ , ] with 15 cycles (Input DNA) or 17 cycles (ChIP DNA) of amplification.

    Techniques: Labeling

    Barcoded adapters perform similarly to standard Illumina adapters and do not crossover to other samples in the same lane . (a) RNA PolII binding profiles from different biological replicates with the same barcode (PolII_Rep1, dark blue; PolII_Rep3, red), with different barcodes (PolII_Rep2, orange) or without barcode (PolII_Rep4, green) strongly overlap. See also Table 3 . Input DNA serves as a reference (light blue). IGB signal tracks of chromosome 5 between 130,000 and 320,000 are shown for each library. A box in the left panel depicts the enlarged section shown in the right panel between positions 298,000 and 309,000 to illustrate the overlap among all PolII signal tracks. (b) Binding profiles from four different libraries pooled and sequenced in the same flowcell lane show very little resemblance. Shown here are the binding profiles for Cse4_Rep2 (dark blue), Ste12_Rep2 (red), PolII_Rep2 (green) and Input_ACGT (light blue). IGB signal tracks of chromosome 12 between 80,000 and 210,000 are shown for each sample. For (a) and (b), axis and scale normalizations are similar to Figure 2 . (c) Left: Rank-rank comparison of target lists between all pairwise barcoded replicates for Cse4, PolII and Ste12. The horizontal axis shows the fraction of the two lists being compared and the vertical axis shows the fraction of those targets that agree between a given pair of target lists. All comparisons show strong agreement, although the rank lists for Cse4 differ more than PolII or Ste12 for the second half of their length. Right: Rank-rank comparison between barcoded replicates from the same factors (averaged over all pairwise comparisons) compared to rank-rank comparisons for barcoded replicates between different factors: PolII_Rep1 (ACGT) vs. Ste12_Rep1 (TGCT) and Cse4_Rep2 (CATT) vs. Ste12_Rep2 (GTAT).

    Journal: BMC Genomics

    Article Title: Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing

    doi: 10.1186/1471-2164-10-37

    Figure Lengend Snippet: Barcoded adapters perform similarly to standard Illumina adapters and do not crossover to other samples in the same lane . (a) RNA PolII binding profiles from different biological replicates with the same barcode (PolII_Rep1, dark blue; PolII_Rep3, red), with different barcodes (PolII_Rep2, orange) or without barcode (PolII_Rep4, green) strongly overlap. See also Table 3 . Input DNA serves as a reference (light blue). IGB signal tracks of chromosome 5 between 130,000 and 320,000 are shown for each library. A box in the left panel depicts the enlarged section shown in the right panel between positions 298,000 and 309,000 to illustrate the overlap among all PolII signal tracks. (b) Binding profiles from four different libraries pooled and sequenced in the same flowcell lane show very little resemblance. Shown here are the binding profiles for Cse4_Rep2 (dark blue), Ste12_Rep2 (red), PolII_Rep2 (green) and Input_ACGT (light blue). IGB signal tracks of chromosome 12 between 80,000 and 210,000 are shown for each sample. For (a) and (b), axis and scale normalizations are similar to Figure 2 . (c) Left: Rank-rank comparison of target lists between all pairwise barcoded replicates for Cse4, PolII and Ste12. The horizontal axis shows the fraction of the two lists being compared and the vertical axis shows the fraction of those targets that agree between a given pair of target lists. All comparisons show strong agreement, although the rank lists for Cse4 differ more than PolII or Ste12 for the second half of their length. Right: Rank-rank comparison between barcoded replicates from the same factors (averaged over all pairwise comparisons) compared to rank-rank comparisons for barcoded replicates between different factors: PolII_Rep1 (ACGT) vs. Ste12_Rep1 (TGCT) and Cse4_Rep2 (CATT) vs. Ste12_Rep2 (GTAT).

    Article Snippet: To amplify the library, PCR was performed using Illumina genomic DNA primer "1.1" and Illumina genomic DNA primer "2.1" as described [ , ] with 15 cycles (Input DNA) or 17 cycles (ChIP DNA) of amplification.

    Techniques: Binding Assay

    Scheme for yeast barcoded ChIP-Seq . (a) Barcoded ChIP-Seq workflow. Ovals depict yeast cells and squares depict proteins. An aliquot of sheared cell lysate is not immunoprecipitated but is otherwise processed normally (green). This DNA, termed input DNA, is a reference sample for ChIP-Seq. Illumina DNA libraries are generated from both ChIP and input DNA samples. In multiplex ChIP-Seq, a barcoded adapter is ligated to an individual DNA sample. The barcode has 3 unique bases followed by a 'T' to anneal with the end-repaired DNA. Four libraries are then pooled together and applied to a single flowcell lane. After sequencing on the Genome Analyzer, reads are separated according to the first four bases and aligned to the yeast genome. Reads are stacked to generate a signal profile and scored against a pool of input DNA to determine significant transcription factor binding sites. (b) The barcode (orange) is located between Illumina adapter sequences (purple) and ChIP or input DNA inserts (black). The sequencing primer (pink) anneals to the adapter sequences and short reads start with the four bases of the barcode (orange) followed by DNA inserts (black). For the sequencing primer and Illumina adapter, oligonucleotide sequences were given by the manufacturer © 2006 Illumina, Inc. All rights reserved.

    Journal: BMC Genomics

    Article Title: Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing

    doi: 10.1186/1471-2164-10-37

    Figure Lengend Snippet: Scheme for yeast barcoded ChIP-Seq . (a) Barcoded ChIP-Seq workflow. Ovals depict yeast cells and squares depict proteins. An aliquot of sheared cell lysate is not immunoprecipitated but is otherwise processed normally (green). This DNA, termed input DNA, is a reference sample for ChIP-Seq. Illumina DNA libraries are generated from both ChIP and input DNA samples. In multiplex ChIP-Seq, a barcoded adapter is ligated to an individual DNA sample. The barcode has 3 unique bases followed by a 'T' to anneal with the end-repaired DNA. Four libraries are then pooled together and applied to a single flowcell lane. After sequencing on the Genome Analyzer, reads are separated according to the first four bases and aligned to the yeast genome. Reads are stacked to generate a signal profile and scored against a pool of input DNA to determine significant transcription factor binding sites. (b) The barcode (orange) is located between Illumina adapter sequences (purple) and ChIP or input DNA inserts (black). The sequencing primer (pink) anneals to the adapter sequences and short reads start with the four bases of the barcode (orange) followed by DNA inserts (black). For the sequencing primer and Illumina adapter, oligonucleotide sequences were given by the manufacturer © 2006 Illumina, Inc. All rights reserved.

    Article Snippet: To amplify the library, PCR was performed using Illumina genomic DNA primer "1.1" and Illumina genomic DNA primer "2.1" as described [ , ] with 15 cycles (Input DNA) or 17 cycles (ChIP DNA) of amplification.

    Techniques: Chromatin Immunoprecipitation, Immunoprecipitation, Generated, Multiplex Assay, Sequencing, Binding Assay

    Sleeping Beauty (SB) -mediated insertion is enhanced by an increased ratio of circular to linear substrates. ( a ) Schematic representation of the strategy used to increase the circular DNA in transduced cells. The lentiviral vector, pLV/FRT-puro-PGK-SBT-FRT, contains a PGK-puro expression cassette within a SB transposon, which is flanked by two Flp recombination target (FRT) sites. The FRT sites enable recombination (and thereby circle formation) in the presence of Flp recombinase. ( b ) FRT circle formation occurs exclusively in the presence of Flp recombinase. HEK-293 cells were transfected with pCMV-Flpe or pUC19 (as a negative control) before transduction with IDLV/FRT-puro-PGK-SBT-FRT (3.2 pg p24 × 10 5 ). Twenty-four hours after transduction HIRT DNA was harvested for quantitative-PCR analysis to determine the amount of total vector DNA, 2-LTR and 1-FRT circles. ( c ) The presence of Flp recombinase increases the number of puro R resistant colonies. HEK-293 cells were transfected with pCMV-Flpe or pUC19 before cotransduction with IDLV/FRT-puro-PGK-SBT-FRT (3.2 pg p24 × 10 5 ), and IDLV/PGK-SB100X or mSB (1.7 pg p24 × 10 6 ) and selected for puromycin resistance. All experiments were performed in triplicates and are presented as mean value ± SD. *Statistical significance, P = 0.024. CMV, cytomegalovirus; IDLV, integrase-defective lentiviral vectors; PGK, 3-phosphoglycerate kinase promoter.

    Journal: Molecular Therapy

    Article Title: Comparative Genomic Integration Profiling of Sleeping Beauty Transposons Mobilized With High Efficacy From Integrase-defective Lentiviral Vectors in Primary Human Cells

    doi: 10.1038/mt.2011.47

    Figure Lengend Snippet: Sleeping Beauty (SB) -mediated insertion is enhanced by an increased ratio of circular to linear substrates. ( a ) Schematic representation of the strategy used to increase the circular DNA in transduced cells. The lentiviral vector, pLV/FRT-puro-PGK-SBT-FRT, contains a PGK-puro expression cassette within a SB transposon, which is flanked by two Flp recombination target (FRT) sites. The FRT sites enable recombination (and thereby circle formation) in the presence of Flp recombinase. ( b ) FRT circle formation occurs exclusively in the presence of Flp recombinase. HEK-293 cells were transfected with pCMV-Flpe or pUC19 (as a negative control) before transduction with IDLV/FRT-puro-PGK-SBT-FRT (3.2 pg p24 × 10 5 ). Twenty-four hours after transduction HIRT DNA was harvested for quantitative-PCR analysis to determine the amount of total vector DNA, 2-LTR and 1-FRT circles. ( c ) The presence of Flp recombinase increases the number of puro R resistant colonies. HEK-293 cells were transfected with pCMV-Flpe or pUC19 before cotransduction with IDLV/FRT-puro-PGK-SBT-FRT (3.2 pg p24 × 10 5 ), and IDLV/PGK-SB100X or mSB (1.7 pg p24 × 10 6 ) and selected for puromycin resistance. All experiments were performed in triplicates and are presented as mean value ± SD. *Statistical significance, P = 0.024. CMV, cytomegalovirus; IDLV, integrase-defective lentiviral vectors; PGK, 3-phosphoglycerate kinase promoter.

    Article Snippet: Finally, primers corresponding to Illumina adapter sequences were used to yield a directional library, in which sequences complementary to the Illumina genomic DNA sequences primer were located upstream to the transposon IRs or the lentiviral LTRs.

    Techniques: Plasmid Preparation, Expressing, Transfection, Negative Control, Transduction, Real-time Polymerase Chain Reaction

    Efficient genomic integration of Sleeping Beauty (SB) transposons from integrase-defective lentiviral vectors (IDLV)-derived substrates. ( a ) Schematic representations of HIV-1/SB hybrid vectors. pLV/RIR-LIR.PGK-puro is a third generation self-inactivating-vector containing a PGK-puro expression cassette flanked upstream by the SB RIR-LIR cassette which is inserted adjacent to the cPPT. The head-to-head orientation of the SB IRs entails that only circularized lentiviral DNA can serve as substrate for SB transposition. pLV/puro-PGK-SBT contains a PGK-puro expression cassette within a SB transposon oriented in the opposite direction of the viral vector. This design enables transposition from both circular and linear lentiviral-derived substrates. ( b ) Reduced transductional titer of vector containing the PGK-puro expression cassette in reverse transcriptional orientation. HEK-293 cells were transduced with serially diluted integration-proficient viral vectors containing the PGK-puro expression cassette in the context of LV/RIR-LIR.PGK-puro and LV/puro-PGK-SBT. Titer calculations based on the number of puromycin-resistant colonies resulted in 3.2 × 10 6 ± 2.1 × 10 5 and 1.3 × 10 6 ± 4.0 × 10 5 colony-forming units (CFU) per ml of the two vectors, respectively. ( c ) Background insertion is reduced with IDLV/puro-PGK-SBT. HEK-293 cells were transduced with IDLV/RIR-LIR.PGK-puro or IDLV/puro-PGK-SBT together with IDLV/PGK-SB100X or IDLV/PGK-mSB and selected for puromycin resistance. Cotransduction with IDLV/PGK-SB100X resulted in a 9- and 23-fold increase for the IDLV/RIR-LIR.PGK-puro and IDLV/puro-PGK-SBT, respectively, relative to cotransduction with IDLV/PGK-mSB. Total number of puromycin-resistant colonies was normalized to the p24 values of the substrate vectors. All experiments were performed in triplicates and are presented as mean value ± SD. cPPT, central polypurine tract; LIR, left inverted repeat; PGK, 3-phosphoglycerate kinase promoter; puro R , puromycin resistance gene; RIR, right inverted repeat; RRE, rev responsive element; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; ψ, packaging signal.

    Journal: Molecular Therapy

    Article Title: Comparative Genomic Integration Profiling of Sleeping Beauty Transposons Mobilized With High Efficacy From Integrase-defective Lentiviral Vectors in Primary Human Cells

    doi: 10.1038/mt.2011.47

    Figure Lengend Snippet: Efficient genomic integration of Sleeping Beauty (SB) transposons from integrase-defective lentiviral vectors (IDLV)-derived substrates. ( a ) Schematic representations of HIV-1/SB hybrid vectors. pLV/RIR-LIR.PGK-puro is a third generation self-inactivating-vector containing a PGK-puro expression cassette flanked upstream by the SB RIR-LIR cassette which is inserted adjacent to the cPPT. The head-to-head orientation of the SB IRs entails that only circularized lentiviral DNA can serve as substrate for SB transposition. pLV/puro-PGK-SBT contains a PGK-puro expression cassette within a SB transposon oriented in the opposite direction of the viral vector. This design enables transposition from both circular and linear lentiviral-derived substrates. ( b ) Reduced transductional titer of vector containing the PGK-puro expression cassette in reverse transcriptional orientation. HEK-293 cells were transduced with serially diluted integration-proficient viral vectors containing the PGK-puro expression cassette in the context of LV/RIR-LIR.PGK-puro and LV/puro-PGK-SBT. Titer calculations based on the number of puromycin-resistant colonies resulted in 3.2 × 10 6 ± 2.1 × 10 5 and 1.3 × 10 6 ± 4.0 × 10 5 colony-forming units (CFU) per ml of the two vectors, respectively. ( c ) Background insertion is reduced with IDLV/puro-PGK-SBT. HEK-293 cells were transduced with IDLV/RIR-LIR.PGK-puro or IDLV/puro-PGK-SBT together with IDLV/PGK-SB100X or IDLV/PGK-mSB and selected for puromycin resistance. Cotransduction with IDLV/PGK-SB100X resulted in a 9- and 23-fold increase for the IDLV/RIR-LIR.PGK-puro and IDLV/puro-PGK-SBT, respectively, relative to cotransduction with IDLV/PGK-mSB. Total number of puromycin-resistant colonies was normalized to the p24 values of the substrate vectors. All experiments were performed in triplicates and are presented as mean value ± SD. cPPT, central polypurine tract; LIR, left inverted repeat; PGK, 3-phosphoglycerate kinase promoter; puro R , puromycin resistance gene; RIR, right inverted repeat; RRE, rev responsive element; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; ψ, packaging signal.

    Article Snippet: Finally, primers corresponding to Illumina adapter sequences were used to yield a directional library, in which sequences complementary to the Illumina genomic DNA sequences primer were located upstream to the transposon IRs or the lentiviral LTRs.

    Techniques: Derivative Assay, Plasmid Preparation, Expressing, Transduction

    Improved insertion of LV-delivered transposons containing the T2 inverted repeats. ( a ) The optimized inverted repeats, T2, improve integrase-defective lentiviral vectors (IDLV)-derived DNA as Sleeping Beauty (SB) transposase substrate twofold. HEK-293 cells were transduced with IDLV/puro-PGK-SBT (1.3 pg p24 × 10 6 ) or IDLV/puro-PGK-SBT2 (1.2 pg p24 × 10 6 ) together with IDLV/PGK-SB100X (2.0 pg p24 × 10 6 ) or IDLV/PGK-mSB (2.0 pg p24 × 10 6 ) and selected for puromycin resistance. ( b ) Transposition of T2-containing SB transposon from IDLV-derived DNA under nonselective conditions. HEK-293 and K562 cells were cotransduced with IDLV/eGFP-PGK-SBT2 (2.0 pg p24 × 10 5 ) and IDLV/PGK-SB100X (6.7 pg p24 × 10 5 ) or IDLV/PGK-mSB (6.7 pg p24 × 10 5 ) and passaged under nonselection condition for 9 (HEK-293) and 16 (K562) days before flow cytometric analysis for enhanced green fluorescent protein (eGFP) expression. Representative flow cytometry plots are shown at left, whereas bar charts representing experiments performed in triplicates are shown at the right. ( c ) Comparison of gene transfer efficiencies obtained with conventional LV vectors and IDLV/SB vectors. Integration-proficient lentiviral vectors (IPLV) were prepared and used to transduce HEK-293 and K562 cells prior to puromycin selection (left panel) and flow cytometric analysis for eGFP expression (right panel), respectively. The dose of IDLV and IPLV was kept constant for all groups. An additional dose of SB100X-encoding IDLV was added in the middle group (IDLV + SB100X). Titers are presented relative to the titer of IDLV/puro-PGK-SBT2 (left panel) and IDLV/eGFP-PGK-SBT2 (right panel) in the absence of SB100X. All experiments were performed in triplicates and are presented as mean value ± SD. PGK, 3-phosphoglycerate kinase promoter.

    Journal: Molecular Therapy

    Article Title: Comparative Genomic Integration Profiling of Sleeping Beauty Transposons Mobilized With High Efficacy From Integrase-defective Lentiviral Vectors in Primary Human Cells

    doi: 10.1038/mt.2011.47

    Figure Lengend Snippet: Improved insertion of LV-delivered transposons containing the T2 inverted repeats. ( a ) The optimized inverted repeats, T2, improve integrase-defective lentiviral vectors (IDLV)-derived DNA as Sleeping Beauty (SB) transposase substrate twofold. HEK-293 cells were transduced with IDLV/puro-PGK-SBT (1.3 pg p24 × 10 6 ) or IDLV/puro-PGK-SBT2 (1.2 pg p24 × 10 6 ) together with IDLV/PGK-SB100X (2.0 pg p24 × 10 6 ) or IDLV/PGK-mSB (2.0 pg p24 × 10 6 ) and selected for puromycin resistance. ( b ) Transposition of T2-containing SB transposon from IDLV-derived DNA under nonselective conditions. HEK-293 and K562 cells were cotransduced with IDLV/eGFP-PGK-SBT2 (2.0 pg p24 × 10 5 ) and IDLV/PGK-SB100X (6.7 pg p24 × 10 5 ) or IDLV/PGK-mSB (6.7 pg p24 × 10 5 ) and passaged under nonselection condition for 9 (HEK-293) and 16 (K562) days before flow cytometric analysis for enhanced green fluorescent protein (eGFP) expression. Representative flow cytometry plots are shown at left, whereas bar charts representing experiments performed in triplicates are shown at the right. ( c ) Comparison of gene transfer efficiencies obtained with conventional LV vectors and IDLV/SB vectors. Integration-proficient lentiviral vectors (IPLV) were prepared and used to transduce HEK-293 and K562 cells prior to puromycin selection (left panel) and flow cytometric analysis for eGFP expression (right panel), respectively. The dose of IDLV and IPLV was kept constant for all groups. An additional dose of SB100X-encoding IDLV was added in the middle group (IDLV + SB100X). Titers are presented relative to the titer of IDLV/puro-PGK-SBT2 (left panel) and IDLV/eGFP-PGK-SBT2 (right panel) in the absence of SB100X. All experiments were performed in triplicates and are presented as mean value ± SD. PGK, 3-phosphoglycerate kinase promoter.

    Article Snippet: Finally, primers corresponding to Illumina adapter sequences were used to yield a directional library, in which sequences complementary to the Illumina genomic DNA sequences primer were located upstream to the transposon IRs or the lentiviral LTRs.

    Techniques: Derivative Assay, Transduction, Flow Cytometry, Expressing, Cytometry, Selection

    Schematic description of the high-throughput SELEX process. ( A ) Protein expression. ( Top ) Proteins are expressed as fusion proteins with SBP-tagged Gaussia -luciferase. ( Bottom ) The GATEWAY recombination cloning system is used to transfer DNA sequences

    Journal: Genome Research

    Article Title: Multiplexed massively parallel SELEX for characterization of human transcription factor binding specificities

    doi: 10.1101/gr.100552.109

    Figure Lengend Snippet: Schematic description of the high-throughput SELEX process. ( A ) Protein expression. ( Top ) Proteins are expressed as fusion proteins with SBP-tagged Gaussia -luciferase. ( Bottom ) The GATEWAY recombination cloning system is used to transfer DNA sequences

    Article Snippet: DNA concentration was determined (Nanodrop-spectrophotometer) and a 3 pM amount was sequenced (Illumina Genome Analyzer mk.2) with a SELEX-sequencing-primer that is identical to the standard Illumina Genomic DNA sequencing primer except that it lacks the 3′ terminal thymine base (Supplemental Table S3).

    Techniques: High Throughput Screening Assay, Expressing, Luciferase, Clone Assay

    Binding profiles. ( A ) Comparison of determined binding-specificity models with previously known data. The left columns indicate the transcription factor analyzed and its DNA-binding domain family. The SELEX cycle from where the model is derived and the

    Journal: Genome Research

    Article Title: Multiplexed massively parallel SELEX for characterization of human transcription factor binding specificities

    doi: 10.1101/gr.100552.109

    Figure Lengend Snippet: Binding profiles. ( A ) Comparison of determined binding-specificity models with previously known data. The left columns indicate the transcription factor analyzed and its DNA-binding domain family. The SELEX cycle from where the model is derived and the

    Article Snippet: DNA concentration was determined (Nanodrop-spectrophotometer) and a 3 pM amount was sequenced (Illumina Genome Analyzer mk.2) with a SELEX-sequencing-primer that is identical to the standard Illumina Genomic DNA sequencing primer except that it lacks the 3′ terminal thymine base (Supplemental Table S3).

    Techniques: Binding Assay, Derivative Assay