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  • 99
    New England Biolabs ultra dna library prep kit
    Ultra Dna Library Prep Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 5305 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher cdna libraries
    Cdna Libraries, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5482 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc dna libraries
    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 Libraries, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 12520 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc nextera xt dna library preparation kit
    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.
    Nextera Xt Dna Library Preparation Kit, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 8497 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Agilent technologies cdna libraries
    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.
    Cdna Libraries, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 94/100, based on 4998 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs nebnext ultra ii dna library prep kit for illumina
    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.
    Nebnext Ultra Ii Dna Library Prep Kit For Illumina, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1667 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa smart cdna library construction kit
    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.
    Smart Cdna Library Construction Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 2787 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc truseq dna pcr free sample preparation kit
    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.
    Truseq Dna Pcr Free Sample Preparation Kit, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 1806 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene cdna libraries
    Northern blot of total RNA isolated from wild-type and p 6H / p 6H tissues (B, brain; L, liver; and T, testis) and hybridized with <t>BS2c</t> probe (corresponding to nucleotides 12661–14606 of the final rjs <t>cDNA</t> contig, which includes part of RLDc and all of the HECT domain). Markers (not shown) indicate this to be a very large transcript, ≥15 kb. Note the expression of the rjs gene in brain and testis of wild type and the absence of transcript in p 6H / p 6H total RNA. The faint high molecular weight band in p 6H / p 6H brain total RNA probably represents nonspecific hybridization since RT-PCR from p 6H / p 6H mRNA was negative with primers after nucleotide 1993.
    Cdna Libraries, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 581 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc prep kit
    Northern blot of total RNA isolated from wild-type and p 6H / p 6H tissues (B, brain; L, liver; and T, testis) and hybridized with <t>BS2c</t> probe (corresponding to nucleotides 12661–14606 of the final rjs <t>cDNA</t> contig, which includes part of RLDc and all of the HECT domain). Markers (not shown) indicate this to be a very large transcript, ≥15 kb. Note the expression of the rjs gene in brain and testis of wild type and the absence of transcript in p 6H / p 6H total RNA. The faint high molecular weight band in p 6H / p 6H brain total RNA probably represents nonspecific hybridization since RT-PCR from p 6H / p 6H mRNA was negative with primers after nucleotide 1993.
    Prep Kit, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 8662 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    prep kit - by Bioz Stars, 2020-11
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    New England Biolabs nebnext dna library prep master mix set for illumina
    Northern blot of total RNA isolated from wild-type and p 6H / p 6H tissues (B, brain; L, liver; and T, testis) and hybridized with <t>BS2c</t> probe (corresponding to nucleotides 12661–14606 of the final rjs <t>cDNA</t> contig, which includes part of RLDc and all of the HECT domain). Markers (not shown) indicate this to be a very large transcript, ≥15 kb. Note the expression of the rjs gene in brain and testis of wild type and the absence of transcript in p 6H / p 6H total RNA. The faint high molecular weight band in p 6H / p 6H brain total RNA probably represents nonspecific hybridization since RT-PCR from p 6H / p 6H mRNA was negative with primers after nucleotide 1993.
    Nebnext Dna Library Prep Master Mix Set For Illumina, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 914 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nebnext dna library prep master mix set for illumina/product/New England Biolabs
    Average 99 stars, based on 914 article reviews
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    nebnext dna library prep master mix set for illumina - by Bioz Stars, 2020-11
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    Image Search Results


    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, 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: 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

    Non-coinciding cDNA-starts are required to map the crosslink sites within Y-tracts. a The cDNA-starts of PTBP1-iCLIP1 and CLIP experiments are plotted around the ends of > 35 nt Y-tracts that are annotated as T-rich or TC-rich low-complexity sequence in the human genome (hg19). cDNAs of PTBP1-iCLIP1 are divided into four length categories: 17–29 nt, 30–34 nt, 35–39 nt and > 39 nt . b Same as ( a ), but using U2AF2-iCLIP and CLIP cDNAs. c Same as ( a ), but showing the positions of cDNA-ends. d Same as ( b ), but showing the positions of cDNA-ends. e Heatmap showing the coverage of PTBP1-binding motifs at the PTBP1-iCLIP1, PTBP1-iCLIP2, PTBP1-eCLIP or PTBP1-irCLIP crosslink clusters that were defined with a 3-nt clustering window. Each row shows the average coverage for 300 clusters of similar length, sorted from shortest to longest clusters. The white line marks the nucleotide preceding the start and the nucleotide following the median end of all clusters that were combined in each row. A colour key for the coverage per nucleotide of the PTBP1-binding motifs is shown on the right

    Journal: Genome Biology

    Article Title: Insights into the design and interpretation of iCLIP experiments

    doi: 10.1186/s13059-016-1130-x

    Figure Lengend Snippet: Non-coinciding cDNA-starts are required to map the crosslink sites within Y-tracts. a The cDNA-starts of PTBP1-iCLIP1 and CLIP experiments are plotted around the ends of > 35 nt Y-tracts that are annotated as T-rich or TC-rich low-complexity sequence in the human genome (hg19). cDNAs of PTBP1-iCLIP1 are divided into four length categories: 17–29 nt, 30–34 nt, 35–39 nt and > 39 nt . b Same as ( a ), but using U2AF2-iCLIP and CLIP cDNAs. c Same as ( a ), but showing the positions of cDNA-ends. d Same as ( b ), but showing the positions of cDNA-ends. e Heatmap showing the coverage of PTBP1-binding motifs at the PTBP1-iCLIP1, PTBP1-iCLIP2, PTBP1-eCLIP or PTBP1-irCLIP crosslink clusters that were defined with a 3-nt clustering window. Each row shows the average coverage for 300 clusters of similar length, sorted from shortest to longest clusters. The white line marks the nucleotide preceding the start and the nucleotide following the median end of all clusters that were combined in each row. A colour key for the coverage per nucleotide of the PTBP1-binding motifs is shown on the right

    Article Snippet: The supernatant was then added to the beads and incubated at 4 °C for 2 h. Afterwards, the beads were washed with IP buffer (10 mM Tris, 150 mM NaCl, 2.5 mM MgCl2 , 1% NP-40), RIPA-S buffer (50 mM Tris, 1 M NaCl, 5 mM EDTA, 2 M urea, 0.5% NP-40, 0.1% SDS, 1% sodium deoxycolate) and PNK buffer before proceeding to the iCLIP protocol for cDNA library preparation and Illumina HiSeq sequencing produced 50 nt sequence reads (Additional file : Figure S1E, F).

    Techniques: Cross-linking Immunoprecipitation, Sequencing, Binding Assay

    A schematic explaining how different extents of cDNA-end constraints affect binding site assignment. a If the iCLIP library contains a broad range of cDNA lengths and unconstrained positions of cDNA-ends, then crosslink sites are identified in an unbiased manner, allowing assignment of the full binding site (RNA map at the bottom). The crosslink sites assigned by cDNA-starts are marked in red bars and a grey bar marks a crosslink site that is incorrectly assigned by a readthrough cDNA. b If cDNA-ends are constrained, most likely as a result of biased RNase cleavage, then the resulting cDNA-starts do not coincide. Nevertheless, if a broad distribution of cDNA lengths is available and the cDNA-ends are placed far enough from the binding site, then crosslink sites can still be identified across the full binding site, allowing correct assignment, as was seen in the case of eIF4A3-iCLIP2 (Fig. 7d ). c If cDNA-ends are constrained to a position very close to the binding site, then those cDNAs that truncate at crosslink sites in the 3′ region of the binding site are too short to be isolated and mapped to the genome. Therefore, crosslink sites are identified only in the 5′ region of the binding site, leading to an overly narrow assignment of binding sites, as was seen in some of the sites identified by eIF4A3-iCLIP1 and eIF4A3-iCLIP2 (Fig. 7c, d ). d If cDNA-ends are constrained and an iCLIP library contains a narrow distribution of cDNA sizes, then cDNA-end constraints lead to an overly narrow assignment of binding regions, as was seen in the case of eIF4A3-iCLIP1 (Fig. 7c )

    Journal: Genome Biology

    Article Title: Insights into the design and interpretation of iCLIP experiments

    doi: 10.1186/s13059-016-1130-x

    Figure Lengend Snippet: A schematic explaining how different extents of cDNA-end constraints affect binding site assignment. a If the iCLIP library contains a broad range of cDNA lengths and unconstrained positions of cDNA-ends, then crosslink sites are identified in an unbiased manner, allowing assignment of the full binding site (RNA map at the bottom). The crosslink sites assigned by cDNA-starts are marked in red bars and a grey bar marks a crosslink site that is incorrectly assigned by a readthrough cDNA. b If cDNA-ends are constrained, most likely as a result of biased RNase cleavage, then the resulting cDNA-starts do not coincide. Nevertheless, if a broad distribution of cDNA lengths is available and the cDNA-ends are placed far enough from the binding site, then crosslink sites can still be identified across the full binding site, allowing correct assignment, as was seen in the case of eIF4A3-iCLIP2 (Fig. 7d ). c If cDNA-ends are constrained to a position very close to the binding site, then those cDNAs that truncate at crosslink sites in the 3′ region of the binding site are too short to be isolated and mapped to the genome. Therefore, crosslink sites are identified only in the 5′ region of the binding site, leading to an overly narrow assignment of binding sites, as was seen in some of the sites identified by eIF4A3-iCLIP1 and eIF4A3-iCLIP2 (Fig. 7c, d ). d If cDNA-ends are constrained and an iCLIP library contains a narrow distribution of cDNA sizes, then cDNA-end constraints lead to an overly narrow assignment of binding regions, as was seen in the case of eIF4A3-iCLIP1 (Fig. 7c )

    Article Snippet: The supernatant was then added to the beads and incubated at 4 °C for 2 h. Afterwards, the beads were washed with IP buffer (10 mM Tris, 150 mM NaCl, 2.5 mM MgCl2 , 1% NP-40), RIPA-S buffer (50 mM Tris, 1 M NaCl, 5 mM EDTA, 2 M urea, 0.5% NP-40, 0.1% SDS, 1% sodium deoxycolate) and PNK buffer before proceeding to the iCLIP protocol for cDNA library preparation and Illumina HiSeq sequencing produced 50 nt sequence reads (Additional file : Figure S1E, F).

    Techniques: Binding Assay, Isolation

    Constrained cDNA-ends affect the cDNA-starts at 3′ splice sites. a The cDNA-starts ( solid lines ) and cDNA-ends ( dotted lines ) of U2AF2-iCLIP are plotted around intron-exon junctions (position 0 being the first nucleotide of the exon). cDNAs are divided into three length categories: 17–29 nt, 30–34 nt and 35-39 nt; the distribution of all cDNAs together is shown in grey . b Same as ( a ), but using only cDNAs that end in the intron. c Same as ( a ), but using only cDNAs that end in the exon. d Same as ( a ), but showing PTBP1-iCLIP1 cDNA-starts ( full lines ) and cDNA-ends ( dotted lines ). e Same as ( a ), but showing PTBP1-iCLIP2 (using 4SU and optimised RNase conditions) cDNA-starts ( full lines ) and cDNA-ends ( dotted lines ). f Same as ( a ), but showing PTBP1-iCLIP3 (omitting 3′ dephosphorylation) cDNA-starts ( full lines ) and cDNA-ends ( dotted lines ). g The composition of genomic nucleotides around iCLIP cDNA-ends from PTBP1-iCLIP1. h Same as ( g ), but showing PTBP1-iCLIP2. i Same as ( g ), but showing PTBP1-iCLIP3. j Proportions of cDNAs that map to introns which contain cDNA-ends at positions overlapping the last two nucleotides of introns. PTBP1-iCLIP1 and PTBP1-iCLIP2 are compared to PTBP1-iCLIP3 iCLIP, which was performed without using PNK to dephosphorylate RNAs in step 2. This enriches for RNAs that contain a 3′ OH, which can occur when they are cleaved at their 3′ end independently of RNase I, such as the 3′ ends of intron lariats

    Journal: Genome Biology

    Article Title: Insights into the design and interpretation of iCLIP experiments

    doi: 10.1186/s13059-016-1130-x

    Figure Lengend Snippet: Constrained cDNA-ends affect the cDNA-starts at 3′ splice sites. a The cDNA-starts ( solid lines ) and cDNA-ends ( dotted lines ) of U2AF2-iCLIP are plotted around intron-exon junctions (position 0 being the first nucleotide of the exon). cDNAs are divided into three length categories: 17–29 nt, 30–34 nt and 35-39 nt; the distribution of all cDNAs together is shown in grey . b Same as ( a ), but using only cDNAs that end in the intron. c Same as ( a ), but using only cDNAs that end in the exon. d Same as ( a ), but showing PTBP1-iCLIP1 cDNA-starts ( full lines ) and cDNA-ends ( dotted lines ). e Same as ( a ), but showing PTBP1-iCLIP2 (using 4SU and optimised RNase conditions) cDNA-starts ( full lines ) and cDNA-ends ( dotted lines ). f Same as ( a ), but showing PTBP1-iCLIP3 (omitting 3′ dephosphorylation) cDNA-starts ( full lines ) and cDNA-ends ( dotted lines ). g The composition of genomic nucleotides around iCLIP cDNA-ends from PTBP1-iCLIP1. h Same as ( g ), but showing PTBP1-iCLIP2. i Same as ( g ), but showing PTBP1-iCLIP3. j Proportions of cDNAs that map to introns which contain cDNA-ends at positions overlapping the last two nucleotides of introns. PTBP1-iCLIP1 and PTBP1-iCLIP2 are compared to PTBP1-iCLIP3 iCLIP, which was performed without using PNK to dephosphorylate RNAs in step 2. This enriches for RNAs that contain a 3′ OH, which can occur when they are cleaved at their 3′ end independently of RNase I, such as the 3′ ends of intron lariats

    Article Snippet: The supernatant was then added to the beads and incubated at 4 °C for 2 h. Afterwards, the beads were washed with IP buffer (10 mM Tris, 150 mM NaCl, 2.5 mM MgCl2 , 1% NP-40), RIPA-S buffer (50 mM Tris, 1 M NaCl, 5 mM EDTA, 2 M urea, 0.5% NP-40, 0.1% SDS, 1% sodium deoxycolate) and PNK buffer before proceeding to the iCLIP protocol for cDNA library preparation and Illumina HiSeq sequencing produced 50 nt sequence reads (Additional file : Figure S1E, F).

    Techniques: De-Phosphorylation Assay

    A broad cDNA length range ameliorates the effects of constrained cDNA-ends. a The cDNA-starts of eIF4A3 iCLIP and CLIP experiments are plotted around the 1000 exon-exon junctions with the highest number of cDNAs. b Same as ( a ), but showing cDNA-ends. c Heatmap showing the position of cDNA-starts in eIF4A3-iCLIP1 around the 1000 exon-exon junctions with the highest number of cDNAs. Junctions are sorted according to their cDNA-end peak position. Each row shows the average of cDNA counts at all junctions with a cDNA-end peak at the indicated position. The values are normalised against the maximum value across all rows. On the right , the arrows mark parts of the figure in which binding site assignment corresponds to the schematic shown in Fig. 8d . Coloured rectangles mark the main region of eIF4A3 crosslinking ( green ), the expected EJC-binding region ( yellow ) and the position of the cDNA-end peak ( blue ). d Same as ( c ), but for eIF4A3-iCLIP2. The arrow in the figure marks the 17 nt minimal distance between cDNA-starts and the expected EJC-binding region that is required for cDNA-starts to be able to identify crosslink sites within the binding site. On the right , the arrows mark sections that correspond to the schematics shown in Fig. 8c , b

    Journal: Genome Biology

    Article Title: Insights into the design and interpretation of iCLIP experiments

    doi: 10.1186/s13059-016-1130-x

    Figure Lengend Snippet: A broad cDNA length range ameliorates the effects of constrained cDNA-ends. a The cDNA-starts of eIF4A3 iCLIP and CLIP experiments are plotted around the 1000 exon-exon junctions with the highest number of cDNAs. b Same as ( a ), but showing cDNA-ends. c Heatmap showing the position of cDNA-starts in eIF4A3-iCLIP1 around the 1000 exon-exon junctions with the highest number of cDNAs. Junctions are sorted according to their cDNA-end peak position. Each row shows the average of cDNA counts at all junctions with a cDNA-end peak at the indicated position. The values are normalised against the maximum value across all rows. On the right , the arrows mark parts of the figure in which binding site assignment corresponds to the schematic shown in Fig. 8d . Coloured rectangles mark the main region of eIF4A3 crosslinking ( green ), the expected EJC-binding region ( yellow ) and the position of the cDNA-end peak ( blue ). d Same as ( c ), but for eIF4A3-iCLIP2. The arrow in the figure marks the 17 nt minimal distance between cDNA-starts and the expected EJC-binding region that is required for cDNA-starts to be able to identify crosslink sites within the binding site. On the right , the arrows mark sections that correspond to the schematics shown in Fig. 8c , b

    Article Snippet: The supernatant was then added to the beads and incubated at 4 °C for 2 h. Afterwards, the beads were washed with IP buffer (10 mM Tris, 150 mM NaCl, 2.5 mM MgCl2 , 1% NP-40), RIPA-S buffer (50 mM Tris, 1 M NaCl, 5 mM EDTA, 2 M urea, 0.5% NP-40, 0.1% SDS, 1% sodium deoxycolate) and PNK buffer before proceeding to the iCLIP protocol for cDNA library preparation and Illumina HiSeq sequencing produced 50 nt sequence reads (Additional file : Figure S1E, F).

    Techniques: Cross-linking Immunoprecipitation, Binding Assay

    An overview of methods and experiments. a A simplified schematic of the iCLIP protocol [ 17 ]. Before, cells or tissues are irradiated with UV light, which creates covalent bonds between proteins and RNAs that are in direct contact (step 1). After lysis, the crosslinked RNA is fragmented by limited concentration of RNase I and RNA fragments are then co-immunoprecipitated with the RBP (step 2), followed by ligation of a 3′ adapter (step 3). After SDS-PAGE purification (step 4), the crosslinked RBP is removed through proteinase K digestion and purification of RNA fragments (step 5). Reverse transcription is performed with a primer that includes a barcode (orange) containing both an experimental identifier and a unique molecular identifier (UMI) (step 6). The peptide that is on the crosslink site impairs reverse transcription and commonly leads to truncation of cDNAs at the crosslink site. Therefore, two types of cDNAs are generated: truncated cDNAs and readthrough cDNAs. In iCLIP, the cDNA library is prepared in such a way that both truncated and readthrough cDNAs are amplified (step 7). After PCR amplification and sequencing (step 8), both truncated and readthrough cDNAs are present. b Table summarising the experiments used in this study. 4SU using 4SU combined with UV-A crosslinking, RNase optimised RNase digest conditions including antiRNase inhibitor and increased RNase I concentration, dephospho omitting 3′ dephosphorylation

    Journal: Genome Biology

    Article Title: Insights into the design and interpretation of iCLIP experiments

    doi: 10.1186/s13059-016-1130-x

    Figure Lengend Snippet: An overview of methods and experiments. a A simplified schematic of the iCLIP protocol [ 17 ]. Before, cells or tissues are irradiated with UV light, which creates covalent bonds between proteins and RNAs that are in direct contact (step 1). After lysis, the crosslinked RNA is fragmented by limited concentration of RNase I and RNA fragments are then co-immunoprecipitated with the RBP (step 2), followed by ligation of a 3′ adapter (step 3). After SDS-PAGE purification (step 4), the crosslinked RBP is removed through proteinase K digestion and purification of RNA fragments (step 5). Reverse transcription is performed with a primer that includes a barcode (orange) containing both an experimental identifier and a unique molecular identifier (UMI) (step 6). The peptide that is on the crosslink site impairs reverse transcription and commonly leads to truncation of cDNAs at the crosslink site. Therefore, two types of cDNAs are generated: truncated cDNAs and readthrough cDNAs. In iCLIP, the cDNA library is prepared in such a way that both truncated and readthrough cDNAs are amplified (step 7). After PCR amplification and sequencing (step 8), both truncated and readthrough cDNAs are present. b Table summarising the experiments used in this study. 4SU using 4SU combined with UV-A crosslinking, RNase optimised RNase digest conditions including antiRNase inhibitor and increased RNase I concentration, dephospho omitting 3′ dephosphorylation

    Article Snippet: The supernatant was then added to the beads and incubated at 4 °C for 2 h. Afterwards, the beads were washed with IP buffer (10 mM Tris, 150 mM NaCl, 2.5 mM MgCl2 , 1% NP-40), RIPA-S buffer (50 mM Tris, 1 M NaCl, 5 mM EDTA, 2 M urea, 0.5% NP-40, 0.1% SDS, 1% sodium deoxycolate) and PNK buffer before proceeding to the iCLIP protocol for cDNA library preparation and Illumina HiSeq sequencing produced 50 nt sequence reads (Additional file : Figure S1E, F).

    Techniques: Irradiation, Lysis, Concentration Assay, Immunoprecipitation, Ligation, SDS Page, Purification, Generated, cDNA Library Assay, Amplification, Polymerase Chain Reaction, Sequencing, De-Phosphorylation Assay

    Crosslink-associated (CL)-motifs are enriched at cDNA deletions and cDNA-starts in iCLIP. a Proportion of eIF4A3 cDNAs with deletion at each position relative to the cDNA-start. Only cDNAs shorter than 40 nt are examined. b Analysis of all PTBP1 experiments examined in this study shows the proportion of cDNAs from each experiment that overlap with a CL-motif at each position relative to the cDNA-start. c Proportion of eIF4A3-CLIP3 cDNAs that overlap with a CL-motif at each position relative to the cDNA-start. Only cDNAs shorter than 40 nt are examined; they are divided into those lacking deletions or containing a deletion within the first 7 nt or anywhere in the remaining portion of the cDNA. d The cDNAs of eIF4A3-CLIP3 containing a deletion within the first 7 nt are further sub-divided into three categories. First, cDNAs with CL-motifs between the 1st and 10th nucleotide of the cDNA. Second, the remaining cDNAs that contain CL-motifs at the position 0. And third, all remaining cDNAs. The proportion of cDNAs that overlap with a CL-motif at each position relative to the cDNA-start is then plotted for each sub-category. e Proportion of PTBP1-iCLIP2 cDNAs that overlap with a CL-motif at each position relative to the cDNA-start. Only cDNAs shorter than 40 nt are examined and are divided into those lacking T-to-C transitions or containing a transition within the first 7 nt or anywhere in the remaining portion of the cDNA. f The cDNAs of PTBP1-iCLIP2 containing a T-to-C transition within the first 7 nt are further sub-divided into three categories. First, cDNAs with CL-motifs overlapping the position 0. Second, the remaining cDNAs that contain CL-motifs between the 1st and 10th nucleotide of the cDNA. And third, all remaining cDNAs. Visualisation as in ( d ). g Same as ( c ), but for PTBP1-iCLIP1. h Same as ( d ), but for PTBP1-iCLIP1

    Journal: Genome Biology

    Article Title: Insights into the design and interpretation of iCLIP experiments

    doi: 10.1186/s13059-016-1130-x

    Figure Lengend Snippet: Crosslink-associated (CL)-motifs are enriched at cDNA deletions and cDNA-starts in iCLIP. a Proportion of eIF4A3 cDNAs with deletion at each position relative to the cDNA-start. Only cDNAs shorter than 40 nt are examined. b Analysis of all PTBP1 experiments examined in this study shows the proportion of cDNAs from each experiment that overlap with a CL-motif at each position relative to the cDNA-start. c Proportion of eIF4A3-CLIP3 cDNAs that overlap with a CL-motif at each position relative to the cDNA-start. Only cDNAs shorter than 40 nt are examined; they are divided into those lacking deletions or containing a deletion within the first 7 nt or anywhere in the remaining portion of the cDNA. d The cDNAs of eIF4A3-CLIP3 containing a deletion within the first 7 nt are further sub-divided into three categories. First, cDNAs with CL-motifs between the 1st and 10th nucleotide of the cDNA. Second, the remaining cDNAs that contain CL-motifs at the position 0. And third, all remaining cDNAs. The proportion of cDNAs that overlap with a CL-motif at each position relative to the cDNA-start is then plotted for each sub-category. e Proportion of PTBP1-iCLIP2 cDNAs that overlap with a CL-motif at each position relative to the cDNA-start. Only cDNAs shorter than 40 nt are examined and are divided into those lacking T-to-C transitions or containing a transition within the first 7 nt or anywhere in the remaining portion of the cDNA. f The cDNAs of PTBP1-iCLIP2 containing a T-to-C transition within the first 7 nt are further sub-divided into three categories. First, cDNAs with CL-motifs overlapping the position 0. Second, the remaining cDNAs that contain CL-motifs between the 1st and 10th nucleotide of the cDNA. And third, all remaining cDNAs. Visualisation as in ( d ). g Same as ( c ), but for PTBP1-iCLIP1. h Same as ( d ), but for PTBP1-iCLIP1

    Article Snippet: The supernatant was then added to the beads and incubated at 4 °C for 2 h. Afterwards, the beads were washed with IP buffer (10 mM Tris, 150 mM NaCl, 2.5 mM MgCl2 , 1% NP-40), RIPA-S buffer (50 mM Tris, 1 M NaCl, 5 mM EDTA, 2 M urea, 0.5% NP-40, 0.1% SDS, 1% sodium deoxycolate) and PNK buffer before proceeding to the iCLIP protocol for cDNA library preparation and Illumina HiSeq sequencing produced 50 nt sequence reads (Additional file : Figure S1E, F).

    Techniques:

    Proportion of non-coinciding cDNA-starts differs between PTBP1 iCLIP experiments. a Heatmap for PTBP1-iCLIP1 generated using the previously developed software iCLIPro [ 8 ] to show the relative positioning of cDNA-starts of shorter iCLIP cDNAs (17–39 nt) compared to cDNA-starts of long cDNAs (longer than 39 nt). b As in ( a ), but for cDNAs of PTBP1-iCLIP1 that overlap with 5–30 nt long crosslink clusters. c As in ( a ), but for cDNAs of PTBP1-iCLIP1 that overlap with > 30 nt long crosslink clusters. d As in ( a ), but for PTBP1-iCLIP2. e As in ( a ), but for cDNAs of PTBP1-iCLIP2 that overlap with 5–30 nt long crosslink clusters. f As in ( a ), but for cDNAs of PTBP1-iCLIP2 that overlap with > 30 nt long crosslink clusters

    Journal: Genome Biology

    Article Title: Insights into the design and interpretation of iCLIP experiments

    doi: 10.1186/s13059-016-1130-x

    Figure Lengend Snippet: Proportion of non-coinciding cDNA-starts differs between PTBP1 iCLIP experiments. a Heatmap for PTBP1-iCLIP1 generated using the previously developed software iCLIPro [ 8 ] to show the relative positioning of cDNA-starts of shorter iCLIP cDNAs (17–39 nt) compared to cDNA-starts of long cDNAs (longer than 39 nt). b As in ( a ), but for cDNAs of PTBP1-iCLIP1 that overlap with 5–30 nt long crosslink clusters. c As in ( a ), but for cDNAs of PTBP1-iCLIP1 that overlap with > 30 nt long crosslink clusters. d As in ( a ), but for PTBP1-iCLIP2. e As in ( a ), but for cDNAs of PTBP1-iCLIP2 that overlap with 5–30 nt long crosslink clusters. f As in ( a ), but for cDNAs of PTBP1-iCLIP2 that overlap with > 30 nt long crosslink clusters

    Article Snippet: The supernatant was then added to the beads and incubated at 4 °C for 2 h. Afterwards, the beads were washed with IP buffer (10 mM Tris, 150 mM NaCl, 2.5 mM MgCl2 , 1% NP-40), RIPA-S buffer (50 mM Tris, 1 M NaCl, 5 mM EDTA, 2 M urea, 0.5% NP-40, 0.1% SDS, 1% sodium deoxycolate) and PNK buffer before proceeding to the iCLIP protocol for cDNA library preparation and Illumina HiSeq sequencing produced 50 nt sequence reads (Additional file : Figure S1E, F).

    Techniques: Generated, Software

    Northern blot of total RNA isolated from wild-type and p 6H / p 6H tissues (B, brain; L, liver; and T, testis) and hybridized with BS2c probe (corresponding to nucleotides 12661–14606 of the final rjs cDNA contig, which includes part of RLDc and all of the HECT domain). Markers (not shown) indicate this to be a very large transcript, ≥15 kb. Note the expression of the rjs gene in brain and testis of wild type and the absence of transcript in p 6H / p 6H total RNA. The faint high molecular weight band in p 6H / p 6H brain total RNA probably represents nonspecific hybridization since RT-PCR from p 6H / p 6H mRNA was negative with primers after nucleotide 1993.

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

    Article Title: A very large protein with diverse functional motifs is deficient in rjs (runty, jerky, sterile) mice

    doi:

    Figure Lengend Snippet: Northern blot of total RNA isolated from wild-type and p 6H / p 6H tissues (B, brain; L, liver; and T, testis) and hybridized with BS2c probe (corresponding to nucleotides 12661–14606 of the final rjs cDNA contig, which includes part of RLDc and all of the HECT domain). Markers (not shown) indicate this to be a very large transcript, ≥15 kb. Note the expression of the rjs gene in brain and testis of wild type and the absence of transcript in p 6H / p 6H total RNA. The faint high molecular weight band in p 6H / p 6H brain total RNA probably represents nonspecific hybridization since RT-PCR from p 6H / p 6H mRNA was negative with primers after nucleotide 1993.

    Article Snippet: After confirmation by Southern and Northern blots, the insert from the largest of these clones (BS2c) was used to screen two additional randomly primed cDNA libraries prepared from testis [gift of T. Miki, National Institutes of Health ( )] and 16.5-day embryos (from Stratagene).

    Techniques: Northern Blot, Isolation, Expressing, Molecular Weight, Hybridization, Reverse Transcription Polymerase Chain Reaction

    Southern blot analysis of genomic DNA from wild type, p 6H / p 6H , p bs / p bs , p cp / p cp , and p 25H / p 25H homozygous mice digested with Hin dIII and hybridized with the BS2c probe (corresponding to nucleotides 12661–14606 of the final rjs cDNA contig that includes part of RLDc and all of the HECT domain). Marker lane contains size markers with position in kb indicated at left. Note deletion of all bands except lowest molecular weight band in p 6H / p 6H DNA.

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

    Article Title: A very large protein with diverse functional motifs is deficient in rjs (runty, jerky, sterile) mice

    doi:

    Figure Lengend Snippet: Southern blot analysis of genomic DNA from wild type, p 6H / p 6H , p bs / p bs , p cp / p cp , and p 25H / p 25H homozygous mice digested with Hin dIII and hybridized with the BS2c probe (corresponding to nucleotides 12661–14606 of the final rjs cDNA contig that includes part of RLDc and all of the HECT domain). Marker lane contains size markers with position in kb indicated at left. Note deletion of all bands except lowest molecular weight band in p 6H / p 6H DNA.

    Article Snippet: After confirmation by Southern and Northern blots, the insert from the largest of these clones (BS2c) was used to screen two additional randomly primed cDNA libraries prepared from testis [gift of T. Miki, National Institutes of Health ( )] and 16.5-day embryos (from Stratagene).

    Techniques: Southern Blot, Mouse Assay, Marker, Molecular Weight