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Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This protocol details how to construct Large Insert libraries from start to finish using NEBNext reagents. The corresponding NEB manual is here: https://www.neb.com/-/media/nebus/files/manuals/manuale7120.pdf and this protocol relates to section 2.OverviewThe Enzymatic Methyl-seq kit (EM-seq) for Illumina contains all the components needed to make libraries that are enzymatically modified to detect 5-methylcytosines (5mC) and 5-hydroxymethylcytosines (5hmC). Figure 1 is an overview of the EM-seq workflow. Firstly, a library is made by ligating EM-seq adaptor to sheared end repaired/dA-tailed genomic DNA. This is followed by two sets of enzymatic conversion steps to differentiate cytosines from 5mC and 5hmC. Finally, libraries are PCR amplified before sequencing.Figure 2 shows a comparison of the sodium bisulfite and EM-seq methods. Sodium bisulfite treatment of DNA results in the deamination of cytosines into uracils, however the modified forms of cytosine (5mC and 5hmC) are not deaminated. Therefore, the preference of bisulfite to chemically deaminate cytosines enables the methylation status of cytosines to be determined. When bisulfite treated DNA is PCR amplified, uracils are replaced by thymines and the 5mC/5hmC are replaced by cytosines. Once sequenced, unmethylated cytosines are represented by thymines and 5mC and 5hmC are represented by cytosines. By comparing sequences to non-converted genomes the appropriate methylation status can be assessed.Enzymatic Methyl-seq is a two step enzymatic conversion process to detect modified cytosines. The first step uses TET2 and an oxidation enhancer to protect modified cytosines from downstream deamination. TET2 enzymatically oxidizes 5mC and 5hmC through a cascade reaction into 5-carboxycytosine [5-methylcytosine (5mC) Þ5-hydroxymethylcytosine (5hmC) Þ 5-formylcytosine (5fC) Þ 5- carboxycytosine (5caC)]. This protects 5mC and 5hmC from deamination. 5hmC can also be protected from deamination by glucosylation to form 5ghmc using the oxidation enhancer. The second enzymatic step uses APOBEC to deaminate C but does not convert 5caC and 5ghmC. The resulting converted sequence can be analyzed like bisulfite-treated DNA. Typical aligners used to analyze data include but are not limited to Bismark and BWAMeth.The workflow described in the NEBNext Enzymatic Methyl-seq Kit is user-friendly and enables methylation detection from inputs ranging between 10 ng–200 ng. EM-seq converted DNA is more intact than bisulfite-converted DNA, resulting in libraries with longer sequencing reads, reduced GC bias and more even genome coverage. Each kit component must pass rigorous quality control standards, and for each new lot the entire set of reagents is functionally validated together by construction of indexed libraries and sequenced on an Illumina sequencing platform. For larger volume requirements, customized and bulk packaging is available by purchasing through the Custom Solutions department at NEB. Please contact [email protected] for further information.

Proper citation: New England Biolabs 2020. Protocol for Use with Large Insert Libraries (470–520 bp) (NEB #E7120). protocols.io https://dx.doi.org/10.17504/protocols.io.bfu2jnye Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: The kit formulation allows for efficient capped RNA synthesis using cap analog (ARCA).

Proper citation: New England Biolabs 2015. Capped RNA Synthesis (E2050). protocols.io https://dx.doi.org/10.17504/protocols.io.cryv7v Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: TEV Protease, also known as Tobacco Etch Virus (TEV) Protease, is a highly specific cysteine protease that recognizes the amino-acid sequence Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser) and cleaves between the Gln and Gly/Ser residues. It is often used for the removal of affinity purification tags such as maltose-binding protein (MBP) or poly-histidine from fusion proteins. TEV Protease has a 7xHis-tag for easy removal from a reaction using nickel affinity resins and has been engineered to improve thermal stability and decrease autolysis. 

Proper citation: New England Biolabs 2020. Cleavage of the Fusion Protein (TEV Protease). protocols.io https://dx.doi.org/10.17504/protocols.io.bfd9ji96 Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This protocols is to be performed with the Quick Ligation Reaction Buffer. Please see the NEB website for more information.

Proper citation: New England Biolabs 2017. Quick Ligation Protocol (M2200). protocols.io https://dx.doi.org/10.17504/protocols.io.iqvcdw6 Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)

Proper citation: New England Biolabs 2020. Protocol for Standard RNA Synthesis with Hi-T7 RNA Polymerase (NEB #M0658). protocols.io https://dx.doi.org/10.17504/protocols.io.8tnhwme Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: Guidelines for Primer Design for Restriction Enzyme Cloning (E6901)

Proper citation: New England Biolabs 2015. Primer Design for Restriction Enzyme Cloning (E6901). protocols.io https://dx.doi.org/10.17504/protocols.io.cpnvmd Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This protocols is for PCR using Q5® High-Fidelity DNA Polymerase (M0491)

Proper citation: New England Biolabs 2015. PCR with Q5® Polymerase (M0491). protocols.io https://dx.doi.org/10.17504/protocols.io.cidua5 Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)

Proper citation: New England Biolabs 2020. Thermolabile Proteinase K Typical Reaction Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.7r6hm9e Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: T7 Endonuclease I recognizes and cleaves non-perfectly matched DNA. This protocol describes how to determine genome targeting efficiency by digesting annealed PCR products with T7 Endonuclease I. In the first step PCR products are produced from the genomic DNA of cells whose genomes were targeted using Cas9, TALEN, ZFN etc. In the second step, the PCR products are annealed and digested with T7 Endonuclease I. Fragments are analyzed to determine the efficiency of genome targeting.

Proper citation: New England Biolabs 2015. Determining Genome Targeting Efficiency using T7 Endonuclease I (M0302). protocols.io https://dx.doi.org/10.17504/protocols.io.cqpvvm Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: The NEBNext Ultra II Directional RNA Library Prep Kit for Illumina contains the enzymes and buffers required to convert a broad range of input amounts of RNA into high quality directional (strand-specific) libraries for next-generation sequencing on the Illumina platform. The fast, user-friendly workflow has minimal hands-on time and is compatible with poly(A) mRNA enrichment and rRNA depletion methods.

Proper citation: New England Biolabs 2018. Protocol for use with FFPE RNA, NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (NEB #E6310) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760, E7765). protocols.io https://dx.doi.org/10.17504/protocols.io.tumenu6 Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: The NEBNext Ultra II FS DNA Library Prep Kit for Illumina contains the enzymes and buffers required to convert a broad range of input amounts of DNA into high quality libraries for next-generation sequencing on the Illumina platform. The fast, user-friendly workflow also has minimal hands-on time.Note: The Ultra II FS Kit is not compatible with bisulfite conversion workflows.

Proper citation: New England Biolabs 2021. E7805 NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina® Protocol for Large Fragment Sizes (> 550 bp) (Chapter 3). protocols.io https://dx.doi.org/10.17504/protocols.io.n2idgce Copy   

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Authors: New England Biolabs, Menna Teffera
Group: New England Biolabs (NEB)
Summary: This module is part of the Ultra™ II workflow, and is optimized for use with the NEBNext®Ultra II End Repair/dA-Tailing Module (NEB #E7546), for Illumina®-compatible library construction.The NEBNext Ultra II Ligation Module is optimized for use with the NEBNext Ultra II End Repair/dA-Tailing Module (NEB #E7546) or the NEBNext Ultra II FS DNA Module (NEB #E7810).

Proper citation: New England Biolabs, Menna Teffera 2019. NEBNext Ultra II Ligation Module (NEB # E7595) for NEBNext Ultra II FS DNA Module (NEB # E7810). protocols.io https://dx.doi.org/10.17504/protocols.io.4ntgven Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This product can be used for the following applications:Cloning of restriction fragmentsJoining linkers and adapters to blunt-ended DNA

Proper citation: New England Biolabs 2021. Ligation Protocol with T4 DNA Ligase (M0202). protocols.io https://dx.doi.org/10.17504/protocols.io.bcchist6 Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: Q5U Hot Start High-Fidelity DNA Polymerase is a modified version of Q5®High-Fidelity DNA Polymerase, a novel thermostable DNA polymerase that possesses 3′ to 5′ exonuclease activity, and is fused to a processivity-enhancing Sso7d domain. Q5U contains a mutation in the uracil-binding pocket that enables the ability to read and amplify templates containing uracil and inosine bases.

Proper citation: New England Biolabs 2020. PCR Using Q5U Hot Start High-Fidelity DNA Polymerase (NEB #M0515): Amplification of bisulfite-converted, deaminated, or damaged DNA (Including FFPE DNA). protocols.io https://dx.doi.org/10.17504/protocols.io.7sdhna6 Copy   

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Authors: New England Biolabs, Menna Teffera
Summary: This module is part of the Ultra™ II workflow, and is optimized for use with the NEBNext®Ultra II End Repair/dA-Tailing Module (NEB #E7546), for Illumina®-compatible library construction.The NEBNext Ultra II Ligation Module is optimized for use with the NEBNext Ultra II End Repair/dA-Tailing Module (NEB #E7546) or the NEBNext Ultra II FS DNA Module (NEB #E7810).

Proper citation: New England Biolabs, Menna Teffera 2019. NEBNext Ultra II Ligation Module (NEB # E7595) for NEBNext Ultra II End Repair/dA Tailing Module (NEB #E7546). protocols.io https://dx.doi.org/10.17504/protocols.io.4nqgvdw Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This is the quick protocol for the Q5® Site-Directed Mutagenesis Kit (E0554)

Proper citation: New England Biolabs 2015. Q5® Site-Directed Mutagenesis Kit Quick Protocol (E0554). protocols.io https://dx.doi.org/10.17504/protocols.io.cpyvpv Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)

Proper citation: New England Biolabs 2015. Adaptor Ligation Mixture (E7370). protocols.io https://dx.doi.org/10.17504/protocols.io.cg6tzd Copy   

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Authors: New England Biolabs
Summary: This is the protocol for the Q5® Site-Directed Mutagenesis Kit (E0554)

Proper citation: New England Biolabs 2020. Q5® Site-Directed Mutagenesis (E0554). protocols.io https://dx.doi.org/10.17504/protocols.io.besrjed6 Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: Radioactive Labeling with T4 Polynucleotide Kinase

Proper citation: New England Biolabs 2015. Radioactive Labeling with T4 PNK (M0201S). protocols.io https://dx.doi.org/10.17504/protocols.io.cp7vrm Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: Cas9 Nuclease, S. pyogenes (Cas9), is a double-stranded DNA endonuclease that is guided to its target by sequence complementarity of a small RNA loaded into the protein. This protocol describes how to digest double-stranded DNA in vitro using Cas9 and a single guide RNA (sgRNA).

Proper citation: New England Biolabs 2014. In vitro digestion of DNA with Cas9 Nuclease, S. pyogenes (M0386). protocols.io https://dx.doi.org/10.17504/protocols.io.ch2t8d Copy   

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