The NEBNext® End Repair Module has been optimized to convert 1 μg – 5μg of fragmented DNA to blunt-

ended DNA having 5´ phosphates, and 3´-hydroxyls. The module is optimized for use with the NEBNext dA-Tailing Module (NEB #E6053), and is part of the original standard DNA library prep workflow.

NEBNext End Repair Enzyme Mix

NEBNext End Repair Reaction Buffer

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The NEBNext® dA-Tailing Module enables incorporation of a non-templated dAMP on the 3´ end of a blunt-ended DNA fragment. The module is optimized for use with the NEBNext dA-Tailing Module (NEB #E6053), and is part of the original standard DNA library prep workflow, which is suitable for 1 – 5 µg of input DNA.

 Klenow Fragment (3´→ 5´ exo–)

 NEBNext dA-Tailing Reaction Buffer

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The NEBNext® Quick Ligation Module has been optimized to ligate efficiently DNA adaptors compatible

with Illumina® sequencing to end-repaired, dA-tailed DNA fragments. The module is optimized for use with the NEBNext End Repair Module (NEB #E6050) and the NEBNext dA-Tailing Module (NEB #E6053) and is part of the original standard DNA library prep workflow, which is suitable for 1 – 5 μg of input DNA.

 Quick T4 DNA Ligase

 NEBNext Quick Ligation Reaction Buffer

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 –  RNA Sample Preparation
 –  Second Strand Synthesis of cDNA

The NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module has been optimized to generate double-stranded cDNA from first-strand cDNA, as part of the NEBNext non-directional RNA library preparation workflow. 

The NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module is Designed for Use with the

Following:

 –  NEBNext Ultra II RNA First Strand Synthesis Module (NEB #E7771)

 –  NEBNext Ultra II End Repair/dA-Tailing Module (NEB #E7546)

 –  NEBNext Ultra II Ligation Module (NEB #E7595)

 –  NEBNext Ultra™ II Q5^® Master Mix (NEB #M0544)

 –  NEBNext^® Oligos for Illumina^® (NEB #E7335, #E7500, #E7710, #E7730, #E6609, #E7600,

    #E7535,  #E7350)

NEBNext^® Second Strand Synthesis Enzyme Mix

NEBNext^® Second Strand Synthesis Reaction Buffer

The NEBNext® Magnesium RNA Fragmentation Module has been optimized to fragment RNA into small

pieces by incubation with Magnesium ions at 94˚C. Fragment size is controlled by incubation time (Figure 1), and the reaction is ended by the addition of NEBNext Fragmentation Stop Solution.

  NEBNext® RNA Fragmentation Buffer

  NEBNext RNA Fragmentation Stop Solution

NEBNext 酶学转化法甲基化建库相关产品

NEBNext 酶学转化法甲基化建库试剂盒

NEBNext 甲基化建库酶学转化法模块

NEBNext Q5U™ 预混液

NEBNext 酶学转化法甲基化建库多样本接头引物（双端检索引物）

虽然重亚硫酸盐测序是研究 DNA 甲基化的金标准，但这种转化方式会对DNA 造成损伤，导致 DNA 断裂、丢失和 GC 偏嗜。NEBNext 酶学转化法甲基化建库试剂盒 (EM-seq^™) 提供了一种酶学方法代替全基因组重亚硫酸盐处理 DNA (WGBS) 的方法，并结合高效流程化的建库步骤，适用于 Illumina 平台测序。高效的 EM-Seq 酶促转化可最大程度地减少对DNA的损伤，结合提供的 NEBNext Ultra II 文库制备流程，最终产生的高质量文库可以从有限的测序数据中更灵敏的检测到 5-mC 和 5-hmC。

更卓越的 5-mC 和 5-hmC 检测灵敏度

更高的比对率

更均一的 GC 覆盖度

能从有限的测序数据检测到更多的 CpG 位点

更均一的二核苷酸分布

更长的文库插入片段

更高效的建库流程

提供转化模块

EM-Seq 能得到更高的文库产量。采用Covaris S2 仪器将 10 ng、50 ng 和 200 ng 不同起始量的人 NA12878 基因组 DNA 打断至 300 bp，同时作为 EM-Seq 和 WGBS 建库的起始样本。对于 WGBS 方法，使用 NEBNext Ultra II DNA 进行建库，随后采用 Zymo Research EZ DNA Methylation-Gold 试剂盒进行重亚硫酸盐转化。上述所有起始量，EM-Seq 都能使用更少的 PCR 循环得到更高的文库产量，表明 EM-Seq 显著减少了 WGBS 方法中的 DNA 损失。误差条表示标准差。

NEBNext 酶学转化法甲基化文库可获得更长的插入片段 。采用Covaris^® S2 仪器将 50 ng 人 NA12878 基因组 DNA 打断至 300 bp，同时作为 EM-Seq 和 WGBS 建库的起始样本。对于 WGBS 方法，使用 NEBNext Ultra II DNA 进行建库，随后采用 Zymo Research EZ DNA Methylation-Gold^™ 试剂盒进行重亚硫酸盐转化。两个文库均使用 Illumina MiSeq (2 X 76 bases) 测序，片段大小采用 Picard 2.18.14 测定。图中绘制了每个插入片段出现的频率标准化后的数据，结果如图：EM-Seq 建库后的插入片段比 WGBS 方法得到的插入片段更长，说明：酶学转化法不会对 DNA 造成损伤，而重亚硫酸盐处理的 DNA 有严重损伤。

A: CpG 位点检测：通过分析 3.24 亿双端数据得到 EM-Seq 和 WGBS 文库的 CpG 位点覆盖度，其中每条链都独立计数，最终得到最多 5600 万个可能的 CpG 位点。结果显示：EM-Seq 在更低测序深度能检测到更多的 CpG 位点。

B: GC 覆盖度：使用 Picard 2.17.2 计算 GC 覆盖度，图中显示不同 GC 含量时 (0-100%)，标准化后覆盖度的分布情况。结果显示：EM-Seq 文库显著提高 GC 覆盖度的均一性，无 AT 过度测序，也无 GC 测序不足，后两项都是 WGBS 文库的典型缺陷。

图中显示了 EM-Seq 和 WGBS 两种方法在不同起始量，至少 1X 和 8X 测序深度下检测到的独有和共有的 CpG 位点。EM-Seq 在至少 1X 测序深度下比 WGBS 多检测到 20% 以上的 CpG 位点。而在至少 8X 测序深度下，CpG 位点覆盖度差异增加至 2 倍。

 停产通知：该产品将于 2021 年 12 月 15 日停产，推荐替代产品为 #E7546S

The NEBNext® Ultra™ End Repair/dA-Tailing Module is optimized to convert 5 ng–1 μg of fragmented DNA to repaired DNA having 5′ phosphorylated, 3’ dA-tailed ends. The module is optimized for use with

NEBNext Ultra Ligation Module (NEB #E7445), and is part of the original Ultra workflow, which enables

library preparation from 5 ng – 1 μg of input DNA in a streamlined protocol.

NEBNext End Prep Enzyme Mix

NEBNext End Repair Reaction Buffer

-20°C

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 停产通知：该产品将于 2021 年 12 月 15 日停产，推荐替代产品为 #E7546S

The NEBNext® Ultra™ End Repair/dA-Tailing Module is optimized to convert 5 ng–1 μg of fragmented DNA to repaired DNA having 5′ phosphorylated, 3’ dA-tailed ends. The module is optimized for use with

NEBNext Ultra Ligation Module (NEB #E7445), and is part of the original Ultra workflow, which enables

library preparation from 5 ng – 1 μg of input DNA in a streamlined protocol. 

    NEBNext End Prep Enzyme Mix

    NEBNext End Repair Reaction Buffer

-20°C

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停产通知：该产品将于 2021 年 12 月 15 日停产，推荐替代产品为 #E7595S

The NEBNext® Ultra™ Ligation Module has been optimized to ligate efficiently DNA adaptors compatible with Illumina® sequencing to end-repaired, dA-tailed DNA fragments. The module is optimized for use with NEBNext Ultra End Repair/dA-Tailing Module (NEB #E7445), and is part of the original Ultra workflow, which achieves library preparation from 5 ng – 1 µg of input DNA in a streamlined protocol. 

The NEBNext Ultra™ Ligation Module is Designed for Use with the Following Products:

NEBNext Ultra™ End Repair/dA-Tailing Module (NEB #E7442)
NEBNext Singleplex or Multiplex Oligos for Illumina® (NEB #E7350, #E7335, #E7500, #E7710 , 

#E7730, #E6609, #E7600)
NEBNext Q5 Hot Start HiFi PCR Master Mix (NEB #M0543)
NEBNext High-Fidelity 2X PCR Master Mix (NEB #M0541)

 Blunt/TA Ligase Master Mix

 NEBNext^® Ligation Enhancer

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– NEBNext Oligo d(T)₂₅ 磁珠

– NEBNext RNA 结合缓冲液（2X）

– NEBNext 洗脱缓冲液

– 无核酸酶污染的水
– NEBNext Tris 缓冲液 

The NEBNext RNA First Strand Synthesis Module is designed for synthesis of cDNA from RNA fragments using ProtoScript® II Reverse Transcriptase and random priming. Single strand cDNA can be used directly for second strand cDNA synthesis. For use in directional (strand-specific) protocols, Actinomycin D is

required and must be purchased separately.

 –  NEBNext First Strand Synthesis Reaction Buffer

 –  Random Primers

 –  ProtoScript® II Reverse Transcriptase

 –  Murine RNase Inhibitor

Materials Required but not Supplied

If you are using the module for Directional RNA Seq, Actinomycin D (Sigma #A1410 dissolved in

dimethylsulfoxide [DMSO] to 5μg/μl) is required. See protocol for details.

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The NEBNext Ultra II End Repair/dA-Tailing Module has been optimized to convert 500 pg-1 μg of fragmented DNA to repaired DNA having 5′ phosphorylated, 3′ dA-tailed ends. The module is optimized for use with the NEBNext Ultra II Ligation Module (NEB #E7595), and is part of the Ultra II DNA workflow which enables high yield preparation of high quality libraries from 500 pg to 1 µg of

input DNA.

This module is compatible with Illumina workflows and with some Oxford Nanopore MinION™ workflows.

For Illumina library construction, the NEBNext Ultra II End Repair/dA-Tailing Module is designed for use with the following:
• NEBNext Ultra II Ligation Module (NEB #E7595)
• NEBNext Ultra II Q5® Master Mix (NEB #M0544)
• NEBNext Oligos for Illumina(NEB #E7335, #E7500, #E7710, #E7730, #E6609, #E7600 #E7535, 

  #E7350)

   NEBNext Ultra II End Prep Enzyme Mix 

   NEBNext Ultra II End Prep Reaction Buffer

-20°C

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The NEBNext^® Ultra^™ II Directional RNA Second Strand Synthesis Module has been optimized to

generate double-stranded cDNA from first-strand cDNA, as part of the NEBNext Directional RNA library

preparation workflow.  This workflow uses the “dUTP” method for strand-specific library construction, which

depends on incorporation of uracil into the second strand cDNA, enabled by the NEBNext Second Strand

Synthesis Reaction Buffer with dUTP Mix, included in this module.

The NEBNext Ultra II Directional RNA Second Strand Synthesis Module is Designed for Use with the

Following:

 –  NEBNext^® Ultra^™ II RNA First Strand Synthesis Module (NEB #E7771)

 –  NEBNext^® Ultra^™ II End Repair/dA-Tailing Module (NEB #E7546)

 –  NEBNext^® Ultra^™ II Ligation Module (NEB #E7595)

 –  NEBNext^® Ultra^™ II Q5^® Master Mix (NEB #M0544)

 –  NEBNext^® Oligos for Illumina^®(NEB #E7335, #E7500, #E7710, #E7730, #E6609, #E7600,         

    #E7535, #E7350)

NEBNext Second Strand Synthesis Enzyme Mix

NEBNext Second Strand Synthesis Reaction Buffer with dUTP Mix

 -20°C

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The NEBNext Ultra II Ligation Module has been optimized to ligate efficiently DNA adaptors compatible

with Illumina sequencing to end-repaired, dA-tailed DNA fragments. The module is optimized for use with

the NEBNext Ultra II End Repair/dA-Tailing Module (NEB #E7546 ), and is part of the Ultra II DNA workflow which enables high yield preparation of high quality libraries from 500 pg to 1 µg of input DNA.

The NEBNext Ultra II Ligation Module is Designed for use with the Following:
 •  NEBNext Ultra II End Repair/dA-Tailing Module (NEB #E7546)
 •  NEBNext Ultra II Q5® Master Mix (NEB #M0544)
 •  NEBNext Oligos for Illumina (NEB #E7335, #E7500, #E7710, #E7730, #E6609, #E7600 #E7535 , 

    NEB #E7350)

    NEBNext® Ligation Enhancer

    NEBNext® Ultra™ II Ligation Master Mix

-20°C 

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 NEBNext® ARTIC SARS-CoV-2 文库制备试剂盒（适用于 Illumina 平台）

目前对 SARS-CoV-2 测序的可靠性和准确性的要求日益增长，为了满足上述需求，NEB 推出 NEBNext® ARTIC 文库制备试剂盒。这些试剂盒根据 ARTIC Network（1）原始流程开发，采用多重扩增子的方法对病毒进行全基因组测序。

SARS-CoV-2 RT-PCR 扩增性能优越。起始样本是 100 ng 人类通用参考 RNA（ThermoFisher QS0639）中的 10 – 10,000 份 SARS-CoV-2 病毒 gRNA 拷贝（ATCC VR-1986 和 VR-1991）。使用 NEBNext 已平衡的 ARTIC SARS-CoV-2 引物池对起始样本进行多重扩增子扩增。使用 Qubit™ dsDNA BR Assay Kit（ThermoFisher Q32850）测定平均扩增子产量。

在 Illumina 平台使用 NEBNext ARTIC SARS-CoV-2 引物池，可提高基因组覆盖度。基因组数据可视化图（IGV）显示了 SARS-CoV-2 基因组的覆盖度。起始样本是 100 ng 人类通用参考 RNA（ThermoFisher QS0639）中的 1,000 份 SARS-CoV-2 病毒 gRNA 拷贝（ATCC VR-1986 和 VR-1991）。分别使用两种引物对起始样本进行多重扩增子扩增：IDT ARTIC nCoV-2019 V3 Panel（“Standard”）和 NEBNext 已平衡的 ARTIC SARS-CoV-2 引物池。建库试剂为 NEBNext ARTIC SARS-CoV-2 FS 文库制备试剂盒（适用于 Illumina 平台）。使用 MiSeq® （2 x 75 bp）测序。测定每个碱基的覆盖深度，向下抽样（seqtk）Reads 数，使用 Bowtie 2 比对至 SARS-CoV-2 参考基因组（NCBI, NC_045512）。

在 Oxford Nanopore Technologies 平台使用 NEBNext ARTIC SARS-CoV-2 引物池，完全覆盖基因组所需的 Reads 数更少。IGV 图显示了 SARS-CoV-2 基因组的覆盖度。使用 IDT ARTIC nCoV-2019 V3 Panel（“Standard”）或 NEBNext 已平衡的 ARTIC SARS-CoV-2 引物池，100 ng 人类通用参考 RNA（ThermoFisher QS0639）中的 1000 份 SARS-CoV-2 病毒 gRNA 拷贝（ATCC VR-1986 和 VR-1991）作为起始样本制备扩增子。使用 NEBNext ARTIC SARS-CoV-2 配套试剂盒（Oxford Nanopore Technologies）、Oxford Nanopore Technologies Native Barcoding Expansion kits 1-12（EXP-NBD104）、1 3-24 (EXP-NBD114)、Ligation Sequencing Kit（EXP-NBD114）、Ligation Sequencing Kit（SQK-LSK109）和 SFB Expansion Kit（EXP-SFB001）制备文库。使用 R9.4.1 flow cells 在 GridION 仪器上进行测序，并使用 Minimap2 比对数据。

NEBNext ARTIC SARS-CoV-2 RT-PCR 模块流程

The NEBNext Ultra II RNA First Strand Synthesis Module has been optimized to convert a broad range of

input amounts of RNA into cDNA using random priming. 

The module is optimized for use with the NEBNext Ultra II Directional RNA Second Strand Synthesis

Module (NEB #E7550) or the NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module

(NEB #E6111), and is part of the Ultra II RNA directional or non-directional workflows. These workflows are compatible with poly(A) mRNA isolation or ribosomal RNA depletion, and enable high yield preparation of

high quality libraries from 5 ng – 1 µg total RNA.

 –  NEBNext First Strand Synthesis Reaction Buffer

 –  Random Primers

 –  NEBNext First Strand Synthesis Enzyme Mix

 –  NEBNext Strand Specificity Reagent

The NEBNext Ultra II FS DNA Module has been optimized to convert 100 pg – 0.5 μg of intact DNA to fragmented, end-repaired DNA having 5´ phosphorylated, 3´ dA-tailed ends. The module is optimized for use

with the NEBNext Ultra II Ligation Module (NEB #E7595), and is part of the Ultra II FS DNA workflow.

For Illumina library construction, the NEBNext Ultra II FS DNA Module is designed for use with the 

following:

• NEBNext Ultra II Ligation Module (NEB #E7595) 
• NEBNext Ultra II Q5® Master Mix (NEB #M0544) 
• NEBNext Oligos for Illumina (NEB #E7335, #E7500, #E7710, #E7730, #E6609, #E7600 #E7535, 
• #E7350) 

TE Buffer

NEBNext® Ultra™ II FS Enzyme Mix

NEBNext® Ultra™ II FS Reaction Buffer

-20°C

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