cip  (New England Biolabs)


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    New England Biolabs cip
    Cip, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cip/product/New England Biolabs
    Average 96 stars, based on 1 article reviews
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    cip - by Bioz Stars, 2022-05
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    New England Biolabs quick calf intestinal phosphatase
    True end-to-end sequencing of single native polyadenylated RNA molecules with 5′ adapter ligation (5TERA). ( A ) Method schematic. Enzymatic treatments to identify indicated 5′ ends by adapter ligation are shown in box. ONT, Oxford Nanopore Technologies; RTA, reverse transcriptase adapter; CIP, <t>Calf</t> <t>Intestinal</t> <t>Phosphatase;</t> RppH, RNA 5′ Pyrophosphohydrolase; 5P, 5′ monophosphate; 5OH, 5′ hydroxyl, Gppp, 5′ cap; A(n), poly(A) tail; T, thymidine. ( B ) Heatmap of read density of the 5′ ends close to the annotated transcription start site based on Ensembl annotation (left) and on re-annotated transcripts (right) from Cap-Poly(A) library. Only molecules with 5′ adapter are used for the analysis. Y-axis corresponds to individual transcripts. Positions up to 150 nucleotides from transcription start site are shown on the x-axis. Z -scores are calculated per row and scale is depicted on top. Number of reads corresponding to each transcript is shown on the right. Only top 30% most expressed transcripts are shown. ( C ) Correlation of the completeness of CDS and mRNA with expression levels based on Ensembl annotation (left) and on re-annotated transcripts (right) from Cap-Poly(A) library. Only molecules with 5′ adapter are used for the analysis. Each point represents an individual transcript. Color represents transcript expression level, calculated as the log2 of reads per million (RPM). Pearson's correlation (R) and associated P -value are shown on top. CDS, Coding Sequence; mRNA, messenger RNA. ( D ) Distribution of molecule ends per transcript length from indicated 5TERA libraries on HeLa re-annotated transcripts. Distribution of reads is calculated for individual transcripts and then averaged for visualizing (green line). Shaded area (green) represents the standard deviation. Only molecules with 5′ adapter are used for the analysis. Meta-coordinates are defined by splitting each transcript into 20 equal bins. Transcript lengths, grouped by 500 nucleotides are shown on the right.
    Quick Calf Intestinal Phosphatase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quick calf intestinal phosphatase/product/New England Biolabs
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    quick calf intestinal phosphatase - by Bioz Stars, 2022-05
    96/100 stars
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    True end-to-end sequencing of single native polyadenylated RNA molecules with 5′ adapter ligation (5TERA). ( A ) Method schematic. Enzymatic treatments to identify indicated 5′ ends by adapter ligation are shown in box. ONT, Oxford Nanopore Technologies; RTA, reverse transcriptase adapter; CIP, Calf Intestinal Phosphatase; RppH, RNA 5′ Pyrophosphohydrolase; 5P, 5′ monophosphate; 5OH, 5′ hydroxyl, Gppp, 5′ cap; A(n), poly(A) tail; T, thymidine. ( B ) Heatmap of read density of the 5′ ends close to the annotated transcription start site based on Ensembl annotation (left) and on re-annotated transcripts (right) from Cap-Poly(A) library. Only molecules with 5′ adapter are used for the analysis. Y-axis corresponds to individual transcripts. Positions up to 150 nucleotides from transcription start site are shown on the x-axis. Z -scores are calculated per row and scale is depicted on top. Number of reads corresponding to each transcript is shown on the right. Only top 30% most expressed transcripts are shown. ( C ) Correlation of the completeness of CDS and mRNA with expression levels based on Ensembl annotation (left) and on re-annotated transcripts (right) from Cap-Poly(A) library. Only molecules with 5′ adapter are used for the analysis. Each point represents an individual transcript. Color represents transcript expression level, calculated as the log2 of reads per million (RPM). Pearson's correlation (R) and associated P -value are shown on top. CDS, Coding Sequence; mRNA, messenger RNA. ( D ) Distribution of molecule ends per transcript length from indicated 5TERA libraries on HeLa re-annotated transcripts. Distribution of reads is calculated for individual transcripts and then averaged for visualizing (green line). Shaded area (green) represents the standard deviation. Only molecules with 5′ adapter are used for the analysis. Meta-coordinates are defined by splitting each transcript into 20 equal bins. Transcript lengths, grouped by 500 nucleotides are shown on the right.

    Journal: Nucleic Acids Research

    Article Title: TERA-Seq: true end-to-end sequencing of native RNA molecules for transcriptome characterization

    doi: 10.1093/nar/gkab713

    Figure Lengend Snippet: True end-to-end sequencing of single native polyadenylated RNA molecules with 5′ adapter ligation (5TERA). ( A ) Method schematic. Enzymatic treatments to identify indicated 5′ ends by adapter ligation are shown in box. ONT, Oxford Nanopore Technologies; RTA, reverse transcriptase adapter; CIP, Calf Intestinal Phosphatase; RppH, RNA 5′ Pyrophosphohydrolase; 5P, 5′ monophosphate; 5OH, 5′ hydroxyl, Gppp, 5′ cap; A(n), poly(A) tail; T, thymidine. ( B ) Heatmap of read density of the 5′ ends close to the annotated transcription start site based on Ensembl annotation (left) and on re-annotated transcripts (right) from Cap-Poly(A) library. Only molecules with 5′ adapter are used for the analysis. Y-axis corresponds to individual transcripts. Positions up to 150 nucleotides from transcription start site are shown on the x-axis. Z -scores are calculated per row and scale is depicted on top. Number of reads corresponding to each transcript is shown on the right. Only top 30% most expressed transcripts are shown. ( C ) Correlation of the completeness of CDS and mRNA with expression levels based on Ensembl annotation (left) and on re-annotated transcripts (right) from Cap-Poly(A) library. Only molecules with 5′ adapter are used for the analysis. Each point represents an individual transcript. Color represents transcript expression level, calculated as the log2 of reads per million (RPM). Pearson's correlation (R) and associated P -value are shown on top. CDS, Coding Sequence; mRNA, messenger RNA. ( D ) Distribution of molecule ends per transcript length from indicated 5TERA libraries on HeLa re-annotated transcripts. Distribution of reads is calculated for individual transcripts and then averaged for visualizing (green line). Shaded area (green) represents the standard deviation. Only molecules with 5′ adapter are used for the analysis. Meta-coordinates are defined by splitting each transcript into 20 equal bins. Transcript lengths, grouped by 500 nucleotides are shown on the right.

    Article Snippet: For selection of only capped RNA molecules, poly(A)-enriched RNA was dephosphorylated on beads using Quick Calf Intestinal Phosphatase (CIP, NEB) according to manufacturer's protocol.

    Techniques: Sequencing, Ligation, Expressing, Standard Deviation

    a. Pie charts representing the proportion of TSS associated with GENCODE genes, predicted tRNAs (orange) or not associated with any annotation (grey) for Pol-II (upper chart) and non-Pol-II (lower chart). b. 7SL (RN7SL1) locus showing the distribution of the 5’ end of mapped reads for Recappable-seq (track 1), CIP treated Recappable-seq (track 2) CAGE (track 3), Pol-II ChIP-seq (track 4, read density) and RNA-seq reads (track 5). The floating panel represents a close up of the 5’ end of the RN7SL1 with two TSS at -1 and +1 of the annotated RN7SL1. The 5’ mapped end of the reads is shown for Recappable-seq (track 1), CIP treated Recappable-seq (track 2), and CAGE (track 3) to mark the TSS positions. c. Validation of the two TSS identified for 7SL using RACE. The amplified fragments were directly sequenced using sanger sequencing with a primer located in the 7SL gene (Material and Methods). The sequencing trace reveals two products ligated to the RACE adaptor resulting from two alternative transcript starts corresponding to TSS1 and TSS2. d. Non-Pol-II TSS flanking tRNA annotations. The top panel visualizes individual non-Pol-II TSS relative to the 5’ end of the annotated mature tRNA (in bp) as a function of the TPM. The bottom panel represents the distribution of the non-Pol-II TSS relative to the start of the annotated tRNA starts (in bp).

    Journal: bioRxiv

    Article Title: ReCappable Seq: Comprehensive Determination of Transcription Start Sites derived from all RNA polymerases

    doi: 10.1101/696559

    Figure Lengend Snippet: a. Pie charts representing the proportion of TSS associated with GENCODE genes, predicted tRNAs (orange) or not associated with any annotation (grey) for Pol-II (upper chart) and non-Pol-II (lower chart). b. 7SL (RN7SL1) locus showing the distribution of the 5’ end of mapped reads for Recappable-seq (track 1), CIP treated Recappable-seq (track 2) CAGE (track 3), Pol-II ChIP-seq (track 4, read density) and RNA-seq reads (track 5). The floating panel represents a close up of the 5’ end of the RN7SL1 with two TSS at -1 and +1 of the annotated RN7SL1. The 5’ mapped end of the reads is shown for Recappable-seq (track 1), CIP treated Recappable-seq (track 2), and CAGE (track 3) to mark the TSS positions. c. Validation of the two TSS identified for 7SL using RACE. The amplified fragments were directly sequenced using sanger sequencing with a primer located in the 7SL gene (Material and Methods). The sequencing trace reveals two products ligated to the RACE adaptor resulting from two alternative transcript starts corresponding to TSS1 and TSS2. d. Non-Pol-II TSS flanking tRNA annotations. The top panel visualizes individual non-Pol-II TSS relative to the 5’ end of the annotated mature tRNA (in bp) as a function of the TPM. The bottom panel represents the distribution of the non-Pol-II TSS relative to the start of the annotated tRNA starts (in bp).

    Article Snippet: ReCappable-seq procedureTotal RNA was (optionally, see text) de-phosphorylated using the Quick CIP (NEB M0525) (3 units/ µgRNA at 0.6 units/µL) and the resulting RNA was purified with the “Clean and concentrate” kit (Zymo Research R1013) using the standard protocol.

    Techniques: Chromatin Immunoprecipitation, RNA Sequencing Assay, Amplification, Sequencing

    ReCappable-seq. a. Principle of ReCappable-seq. 1. RNA is subjected to decapping with yDcpS which acts on capped transcripts originating from Pol-II transcription. Subsequently, the RNA is capped with a biotin-modified GTP analog (3’-desthiobiotin-GTP) using VCE. This biotinylation step allows enrichment of all primary transcripts on a streptavidin matrix. 2. Differentiation of Pol-II from the non-Pol-II transcripts is accomplished by sequencing a second library constructed with RNA treated with CIP prior to the yDcpS treatment, in order to remove the 5’ triphosphate from non-Pol-II transcripts.b. RT-qPCR assay measuring the recovery after streptavidin enrichment of various classes of transcripts such as 18S rRNA as an example of a processed transcript (rRNA), ACTB, RPL19, MALAT (MALAT1), FKBP5, TMSB10, H3H (HIST1H3H) and H3B (HIST1H3B) as examples of capped transcripts, RMRP, RPPH1 and 7SK (RN7SK) as examples of Pol III transcripts (with 7SK having a 5’ methylated triphosphate and therefore resistant to CIP treatment, see main text) and ERCC190 and FLUC-ppp as examples of spiked-in in vitro transcripts with a defined triphosphorylated 5’ end. c. Example of a Pol-II TSS in the TMSB10 locus: the same positions (shaded in pink) are found in the CAGE dataset. CIP treatment intensifies the signal, consistent with a Pol-II TSS. d. Example of Pol-III TSS corresponding to the start of two vault RNAs (vault RNA 1-1 and vault RNA 1-2) located on Chr.5. The positions (shaded in pink) are missing in the CAGE dataset. CIP treatment reduces the signal, consistent with non-Pol-II TSS. In c. and d. the tracks correspond to ReCappable-seq, CIP-Recappable-seq, CAGE, and RNA-seq (A549 rRNA-depleted RNA-seq) read coverage. The four bottom tracks correspond to read density from public ENCODE DNase-seq from A549 cells (ENCFF473YHH, ENCFF809KIH, ENCFF821UUL, ENCFF961WXW).

    Journal: bioRxiv

    Article Title: ReCappable Seq: Comprehensive Determination of Transcription Start Sites derived from all RNA polymerases

    doi: 10.1101/696559

    Figure Lengend Snippet: ReCappable-seq. a. Principle of ReCappable-seq. 1. RNA is subjected to decapping with yDcpS which acts on capped transcripts originating from Pol-II transcription. Subsequently, the RNA is capped with a biotin-modified GTP analog (3’-desthiobiotin-GTP) using VCE. This biotinylation step allows enrichment of all primary transcripts on a streptavidin matrix. 2. Differentiation of Pol-II from the non-Pol-II transcripts is accomplished by sequencing a second library constructed with RNA treated with CIP prior to the yDcpS treatment, in order to remove the 5’ triphosphate from non-Pol-II transcripts.b. RT-qPCR assay measuring the recovery after streptavidin enrichment of various classes of transcripts such as 18S rRNA as an example of a processed transcript (rRNA), ACTB, RPL19, MALAT (MALAT1), FKBP5, TMSB10, H3H (HIST1H3H) and H3B (HIST1H3B) as examples of capped transcripts, RMRP, RPPH1 and 7SK (RN7SK) as examples of Pol III transcripts (with 7SK having a 5’ methylated triphosphate and therefore resistant to CIP treatment, see main text) and ERCC190 and FLUC-ppp as examples of spiked-in in vitro transcripts with a defined triphosphorylated 5’ end. c. Example of a Pol-II TSS in the TMSB10 locus: the same positions (shaded in pink) are found in the CAGE dataset. CIP treatment intensifies the signal, consistent with a Pol-II TSS. d. Example of Pol-III TSS corresponding to the start of two vault RNAs (vault RNA 1-1 and vault RNA 1-2) located on Chr.5. The positions (shaded in pink) are missing in the CAGE dataset. CIP treatment reduces the signal, consistent with non-Pol-II TSS. In c. and d. the tracks correspond to ReCappable-seq, CIP-Recappable-seq, CAGE, and RNA-seq (A549 rRNA-depleted RNA-seq) read coverage. The four bottom tracks correspond to read density from public ENCODE DNase-seq from A549 cells (ENCFF473YHH, ENCFF809KIH, ENCFF821UUL, ENCFF961WXW).

    Article Snippet: ReCappable-seq procedureTotal RNA was (optionally, see text) de-phosphorylated using the Quick CIP (NEB M0525) (3 units/ µgRNA at 0.6 units/µL) and the resulting RNA was purified with the “Clean and concentrate” kit (Zymo Research R1013) using the standard protocol.

    Techniques: Modification, Sequencing, Construct, Quantitative RT-PCR, Methylation, In Vitro, RNA Sequencing Assay

    Library preparation of the targeted enrichment protocol. A schematic overview of the library preparation to enrich a target region (blue). Freshly isolated HMW genomic DNA was dephosphorylated by incubation with CIP for 20 min at 37°C. Subsequently, selected pools of RNPs ( Supplementary Tables 1, 2 ) were added to aliquots of the dephosphorylated gDNA, and incubated for 60 min at 37°C in the presence of Taq polymerase and dATP, followed by incubation for 5 min at 72°C. The available phosphate groups at the terminus of the targeted region were ligated to Nanopore adaptors during incubation with T4 ligase for 60 min at room temperature (RT). The adapter-ligated aliquots were pooled, followed by clean-up with 0.3X AMPure beads. The eluted sample was loaded on a flowcell for Nanopore sequencing.

    Journal: Frontiers in Immunology

    Article Title: Rapid Characterization of Complex Killer Cell Immunoglobulin-Like Receptor (KIR) Regions Using Cas9 Enrichment and Nanopore Sequencing

    doi: 10.3389/fimmu.2021.722181

    Figure Lengend Snippet: Library preparation of the targeted enrichment protocol. A schematic overview of the library preparation to enrich a target region (blue). Freshly isolated HMW genomic DNA was dephosphorylated by incubation with CIP for 20 min at 37°C. Subsequently, selected pools of RNPs ( Supplementary Tables 1, 2 ) were added to aliquots of the dephosphorylated gDNA, and incubated for 60 min at 37°C in the presence of Taq polymerase and dATP, followed by incubation for 5 min at 72°C. The available phosphate groups at the terminus of the targeted region were ligated to Nanopore adaptors during incubation with T4 ligase for 60 min at room temperature (RT). The adapter-ligated aliquots were pooled, followed by clean-up with 0.3X AMPure beads. The eluted sample was loaded on a flowcell for Nanopore sequencing.

    Article Snippet: Input gDNA (5-10 μg) was resuspended in 10x NEB CutSmart Buffer (8:1) and dephosphorylated by incubation with Quick calf intestinal phosphatase (CIP) (NEB, # M0525S) at 37°C for 20 min, followed by heating at 80°C for 2 min to deactivate the enzyme ( ).

    Techniques: Isolation, Incubation, Nanopore Sequencing

    Affinity-based Cas9-Mediated Enrichment (ACME) Workflow. ACME aliows for the targeting of multiple regions of interest within the same reaction with single target read lengths as large as 1Oūkb. For ACME, individual samples starting with 5μg HMW DNA are prepped as follows: - Dephosphorylation: 5’ ends of DNA are dephosphorylated to prevent the ligation of sequencing adapters to non-target strands. - Cas9 binding and cleavage: Target specific crRNAs are bound with tracrRNA to form Cas9 RNPs. These RNPs cleave at either end of the targets, revealing blunt ends with now ligatable 5’ phosphates. After cleavage, the Cas9 enzyme stays bound to the PAM distal end on the non-target flanks, protecting those 5’ ends from adapter ligation. - dA-tailing: All 3’ ends of DNA are dA-tailed to prepare the blunt ends for sequencing adapter ligation. - HisTag isolation and pulldown: The Cas9 enzyme used for cleavage has a 6-Histidine tag at its C-terminal end (inset). Using His-Tag Dynabeads™ we isolate and pulldown Cas9 and the non-target DNA bound to it, thereby reducing background non-target reads. - Adapter ligation and sequencing: Adapters are ligated to either side of the targets and the resultant Cas9 enriched library is loaded onto the flowcell for sequencing.

    Journal: bioRxiv

    Article Title: ACME: an Affinity-based Cas9 Mediated Enrichment method for targeted nanopore sequencing

    doi: 10.1101/2022.02.03.478550

    Figure Lengend Snippet: Affinity-based Cas9-Mediated Enrichment (ACME) Workflow. ACME aliows for the targeting of multiple regions of interest within the same reaction with single target read lengths as large as 1Oūkb. For ACME, individual samples starting with 5μg HMW DNA are prepped as follows: - Dephosphorylation: 5’ ends of DNA are dephosphorylated to prevent the ligation of sequencing adapters to non-target strands. - Cas9 binding and cleavage: Target specific crRNAs are bound with tracrRNA to form Cas9 RNPs. These RNPs cleave at either end of the targets, revealing blunt ends with now ligatable 5’ phosphates. After cleavage, the Cas9 enzyme stays bound to the PAM distal end on the non-target flanks, protecting those 5’ ends from adapter ligation. - dA-tailing: All 3’ ends of DNA are dA-tailed to prepare the blunt ends for sequencing adapter ligation. - HisTag isolation and pulldown: The Cas9 enzyme used for cleavage has a 6-Histidine tag at its C-terminal end (inset). Using His-Tag Dynabeads™ we isolate and pulldown Cas9 and the non-target DNA bound to it, thereby reducing background non-target reads. - Adapter ligation and sequencing: Adapters are ligated to either side of the targets and the resultant Cas9 enriched library is loaded onto the flowcell for sequencing.

    Article Snippet: Genomic DNA was dephosphorylated by adding 3 μl of Quick CIP enzyme (NEB, Cat #M0508) to 5 μg of input DNA (in total volume of 24 μl at > 210 ng/μl) with 3 μl 10X CutSmart Buffer (NEB, Cat #B7204), and incubating for 10 min at 37°C, followed by heating for 2 min at 80°C.

    Techniques: De-Phosphorylation Assay, Ligation, Sequencing, Binding Assay, Isolation