Nebnext ultra ligation module

Manufactured by New England Biolabs

Sourced in United States

The NEBNext Ultra Ligation Module is a laboratory product designed to facilitate the ligation step in next-generation sequencing library preparation. It provides the necessary components, including a highly efficient T4 DNA ligase, to join DNA fragments with compatible ends.

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Lab products found in correlation

Nebnext end repair da tailing module, by New England Biolabs (8 mentions) Nebnext ultra end repair da tailing module, by New England Biolabs (7 mentions) Nextseq 500, by Illumina (4 mentions) Hiseq 2000, by Illumina (4 mentions) Kapa real time amplification kit, by Roche (3 mentions) Ab8580, by Abcam (2 mentions) Ab4729, by Abcam (2 mentions) Agencourt ampure xp bead, by Beckman Coulter (2 mentions) Nebnext ultra 2 dna library prep kit, by Illumina (2 mentions) Hiseq 2500 system, by Illumina (2 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions) Proteinase k, by Thermo Fisher Scientific (2 mentions) Qubit dsdna hs assay, by Thermo Fisher Scientific (2 mentions) Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions) S200 sonicator, by Covaris (1 mentions) Nucleospin tissue kit, by Macherey-Nagel (1 mentions) Ab70303, by Abcam (1 mentions) Protein a g magnetic beads, by Merck Group (1 mentions) Anti androgen receptor antibody, by Merck Group (1 mentions) Gel extraction kit, by Qiagen (1 mentions)

22 protocols using nebnext ultra ligation module

GUIDE-seq analysis of CRISPR off-target effects

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GUIDE-seq experiments were performed as previously described in ref. ^{18 (link)}. Briefly, 2 × 10⁵ HEK293T cells were transfected using Lipofectamine 3000 transfection reagent (Invitrogen) with 1 µg of all-in-one pX plasmid, expressing CoCas9 and sgRNA, and 10 pmol of dsODNs; scramble sgRNA was used as negative control. The day after transfection, cells were detached and selected with 1 µg/ml puromycin as described in ref. ^{19 (link)} Three days after transfection, cells were collected, and genomic DNA extracted using NucleoSpin Tissue Kit (Macherey-Nagel) following manufacturer’s instructions. Using Covaris S200 sonicator, genomic DNA was sheared to an average length of 500 bp. End-repair reaction was performed using NEBNext Ultra End Repair/dA Tailing Module and adaptor ligation using NEBNext® Ultra™ Ligation Module, as described by ref. ^{56 (link)} Amplification steps were then performed following the GUIDE-seq original protocol^{18 (link)}.
Visualization of aligned off-target sites is available as a color-coded sequence grid (Supplementary Fig. 7a–d and Supplementary Fig. 9d–h). GUIDE-seq data are provided as Source data files.

Pedrazzoli E., Demozzi M., Visentin E., Ciciani M., Bonuzzi I., Pezzè L., Lucchetta L., Maule G., Amistadi S., Esposito F., Lupo M., Miccio A., Auricchio A., Casini A., Segata N, & Cereseto A. (2024). CoCas9 is a compact nuclease from the human microbiome for efficient and precise genome editing. Nature Communications, 15, 3478.

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ChIP-seq Library Preparation Protocol

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The preparation of ChIP and input DNA libraries was performed as previously described [32 (link)]. In brief, two cells were crosslinked with 1% formaldehyde for 5 min at room temperature and quenched with glycine (125 mM). Cells were then put on ice, resuspended in cold cell lysis buffer [140 mM NaCl, 1 mM EDTA pH 8.0, 1% Triton X-100, 0.1% SDS, and protease inhibitors (Roche)]. Nuclei were sonicated into fragments of 200–1000 bp in size. The chromatin fragments were precleared and then immunoprecipitated with Protein A + G magnetic beads coupled with anti-H3K4me3 (ab8580; Abcam), anti-H3K27ac (ab4729; Abcam), anti-H3K27me3 (07-449; Millipore), and anti-CTCF (ab70303; Abcam). After reverse crosslinking, immunoprecipitated DNA and input DNA were end-repaired and adapters were ligated to the DNA fragments using NEBNext Ultra End-Repair/dA-Tailing Module (E7442; NEB) and NEBNext Ultra Ligation Module (E7445; NEB). High-throughput sequencing of the ChIP fragments was performed using Illumina NextSeq 500, following the manufacturer’s protocol.

Tian G.G., Zhao X., Hou C., Xie W., Li X., Wang Y., Wang L., Li H., Zhao X., Li J, & Wu J. (2022). Integrative analysis of the 3D genome structure reveals that CTCF maintains the properties of mouse female germline stem cells. Cellular and Molecular Life Sciences, 79(1), 22.

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ChIP-seq Analysis of AR and WT1 in Sertoli Cells

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To characterize genome-wide binding patterns of AR and WT1 in Sertoli cells, ChIP and input DNA libraries were performed as previously described [33 (link)]. Briefly, cells were cross-linked with 1% formaldehyde for 10 min at room temperature and formaldehyde was then inactivated by the addition of 125 mM glycine for 5 min. Sonicated DNA fragments with 100–300 bp were pre-cleared and then immunoprecipitated with Protein A + G Magnetic beads coupled with anti-Androgen Receptor antibody (Millipore, 06–680) or anti-Wilms’ tumor 1 antibody (Abcam, ab89901). After reverse crosslinking, immunoprecipitated DNAs and input DNAs were end-repaired and ligated adapters to the DNA fragments using NEBNext Ultra End-Repair/dA-Tailing Module (E7442, NEB) and NEBNext Ultra Ligation Module (E7445, NEB). High-throughput sequencing of the ChIP fragments was performed using Illumina NextSeq 500 following the manufacturer’s protocols. The raw sequencing data were processed with trimmomatic (version 0.36) to filter low-quality reads [34 (link)]. The resulting data were mapped using bowtie2 (version 2.2.9) to the UCSC mm10 genome reference [35 ]. Peak detection was performed using the MACS peak finding algorithm (Model-based Analysis of ChIP-Seq; version 1.4.2) with parameters --nomodel --shiftsize 25 and the p-value cutoff set to 0.05 [36 (link)].

Wang J., Li J., Xu W., Xia Q., Gu Y., Song W., Zhang X., Yang Y., Wang W., Li H, & Zou K. (2019). Androgen promotes differentiation of PLZF+ spermatogonia pool via indirect regulatory pattern. Cell Communication and Signaling : CCS, 17, 57.

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ChIP-Seq Library Preparation and Sequencing

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For library, 40~50 ng immunoprecipitated DNAs and input DNAs were end-repaired and ligated adapters to the DNA fragments using NEBNext Ultra End-Repair/dA-Tailing Module and NEBNext Ultra Ligation Module (NEB, USA), respectively. DNA size selection was performed using 2% Agarose Gel; then 200~500 bp DNA fragments were excised and purified using Qiagen Gel Extraction Kit (Qiagen, Germany). Each sample was amplified for 14 cycles in a DNA thermal cycler using Q5 High-Fidelity DNA Polymerase (NEB, USA) and corresponding PCR Master Mix. Lastly, the PCR products were quantified using Nanodrop and performed standard single end sequencing with 50 bp reads using Illumina Hiseq 2000 (Illumina, USA). The raw sequencing data of this study are available in the EMBL database under accession number E-MTAB-3992: http://www.ebi.ac.uk/arrayexpress/.

Lu J., Zhang X., Shen T., Ma C., Wu J., Kong H., Tian J., Shao Z., Zhao X, & Xu L. (2016). Epigenetic Profiling of H3K4Me3 Reveals Herbal Medicine Jinfukang-Induced Epigenetic Alteration Is Involved in Anti-Lung Cancer Activity. Evidence-based Complementary and Alternative Medicine : eCAM, 2016, 7276161.

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ChIP-Seq Protocol for Protein-DNA Interactions

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Cells were crosslinked with 1% formaldehyde for 10 min at room temperature and quenched with 125 mM glycine. The fragmented chromatin fragments were precleared and then immunoprecipitated with Protein A+G Magnetic Beads coupled with anti-Flag antibodies. After reverse crosslinking, ChIP and input DNA fragments were end-repaired and A-tailed using the NEBNext End Repair/dA-Tailing Module (E7442, NEB), followed by adapter ligation with the NEBNext Ultra Ligation Module (E7445, NEB). The DNA libraries were amplified for 15 cycles and sequenced using Illumina NovaSeq PE150 as the sequencing mode.

Jing Y.J., Lin L.C., Chen L.L., Huang Z.E., Qin H.C., Li S.B, & Chen Z.H. (2022). WT1 Inhibits Human Renal Carcinoma Cell Proliferation and Induces G2/M Arrest by Upregulating IL-24 Expression. BioMed Research International, 2022, 1093945.

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Microtissue MeDIP-Seq Library Preparation

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For preparation of microtissue MeDIP-Seq libraries, a previously published low input MeDIP protocol^{78 (link)} was modified. DNA was fragmented to 100–200 bp using the Covaris S2 system. Because of low DNA yield for DOX and IDA samples, the triplicate samples were pooled before fragmentation. End repair and A tailing was performed using the NEBNext^® Ultra™ library prep kit for Illumina^® (NEB), adapters were ligated with NEBNext^® Ultra™ Ligation Module (NEB) and samples were purified using Agencourt^® AMPure^® XP beads (Beckman Coulter). Methylated fragments were captured using the MagMeDIP kit (Diagenode). In short, denatured DNA was mixed with anti-5-meC-antibody and captured using magnetic beads. Capture efficiency was determined by qPCR against spiked-in Lambda-DNA fragments in precapture and postcapture library samples. Libraries were amplified in a final PCR step using barcoded TruSeq primers. Quality was assessed on Agilent Bioanalyzer 2100 and library concentration was determined by Qubit™ and qPCR.

Selevsek N., Caiment F., Nudischer R., Gmuender H., Agarkova I., Atkinson F.L., Bachmann I., Baier V., Barel G., Bauer C., Boerno S., Bosc N., Clayton O., Cordes H., Deeb S., Gotta S., Guye P., Hersey A., Hunter F.M., Kunz L., Lewalle A., Lienhard M., Merken J., Minguet J., Oliveira B., Pluess C., Sarkans U., Schrooders Y., Schuchhardt J., Smit I., Thiel C., Timmermann B., Verheijen M., Wittenberger T., Wolski W., Zerck A., Heymans S., Kuepfer L., Roth A., Schlapbach R., Niederer S., Herwig R, & Kleinjans J. (2020). Network integration and modelling of dynamic drug responses at multi-omics levels. Communications Biology, 3, 573.

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ChIP-seq library preparation protocol

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The preparation of ChIP and input DNA libraries were performed as previously described [27 (link)]. Briefly, cells were crosslinked with 1 % formaldehyde for 10 min at room temperature and quenched with 125 mM glycine. The fragmented chromatin fragments were pre-cleared and then immunoprecipitated with Protein A + G Magnetic beads coupled with anti-H3K4me1 (ab8895, Abcam), anti-H3K4me3 (ab8580, Abcam), anti-H3K27ac (ab4729, Abcam), anti-H3K27me3 (07–449, Millipore), and anti-RNA Pol II (ab5131, Abcam) antibodies. After reverse crosslinking, ChIP and input DNA fragments were end-repaired and A-tailed using the NEBNext End Repair/dA-Tailing Module (E7442, NEB) followed by adaptor ligation with the NEBNext Ultra Ligation Module (E7445, NEB). The DNA libraries were amplified for 15 cycles and subjected to deep sequencing with an Illumina HiSeq 2000.

Zhang X.L., Wu J., Wang J., Shen T., Li H., Lu J., Gu Y., Kang Y., Wong C.H., Ngan C.Y., Shao Z., Wu J, & Zhao X. (2016). Integrative epigenomic analysis reveals unique epigenetic signatures involved in unipotency of mouse female germline stem cells. Genome Biology, 17, 162.

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In Situ Hi-C Protocol for Chromatin Conformation Capture

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In situ Hi-C was performed as previously described^{9 (link)} with slight modifications. Cells at 80% confluence in 10 cm dish were fixed in 1% pFA at room temperature for 10 min and subjected to the in situ Hi-C procedure. After proximal ligation, ligation products were enriched by centrifugation at 10,000 rpm at 4 °C for 10 min. The pellet was suspended with proteinase K solution consisting of 10 mM Tris-HCl, pH 8.0, 50 mM EDTA, and 2 mg/ml proteinase K (Thermo Fisher Scientific) and incubated at 37 °C for 1 h, followed by overnight incubation at 68 °C with 0.5 M NaCl. DNA was purified by phenol/chloroform extraction and sonicated at 4 °C using Bioruptor (Diagenode) for 15 min by repeating the cycle of 30-sec ON and 1-min OFF. Biotin-labeled DNA was purified using Dynabeads (MyOne Streptavidin T1, Thermo Fisher Scientific). Sequencing adapters were ligated using NEBNext Ultra Ligation module (New England Biolabs, Inc) on Dynabeads, and DNA was PCR-amplified for 8–14 cycles using NEBNext Q5 Hot Start HiFi PCR master Mix and NEBNext multiplex oligos (New England Biolabs, Inc). PCR products were sequenced on Illumina NextSeq 500 platform to obtain 76-bp paired-end reads.

Iwasaki O., Tanizawa H., Kim K.D., Kossenkov A., Nacarelli T., Tashiro S., Majumdar S., Showe L.C., Zhang R, & Noma K.I. (2019). Involvement of condensin in cellular senescence through gene regulation and compartmental reorganization. Nature Communications, 10, 5688.

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ChIP-seq of SMRC1, SATB1, and NFIC

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The ChIP and input DNA libraries were prepared as previously described (34 (link), 57 (link)). Briefly, 10 million HepG2 cells were cross-linked with 1% formaldehyde for 10 min at room temperature and then quenched with 125 mM glycine. The chromatin was fragmented and then immunoprecipitated with Protein A + G magnetic beads coupled with antibodies against SMRC1, SATB1, and NFIC. After reverse cross-linking, ChIP and input DNA fragments were used for library construction with NEBNext Ultra Ligation Module (NEB, no. E7445). The DNA libraries were amplified and subjected to deep sequencing with an Illumina sequencer. The ChIP-seq data processing was performed as we reported recently (57 (link)). Cis-regulatory sequence elements that mediate the binding of SMRC1, SATB1, or NFIC were predicted with MEME-ChIP (58 (link)).

Wang Z., Li Y., Hou B., Pronobis M.I., Wang M., Wang Y., Cheng G., Weng W., Wang Y., Tang Y., Xu X., Pan R., Lin F., Wang N., Chen Z., Wang S., Ma L.Z., Li Y., Huang D., Jiang L., Wang Z., Zeng W., Zhang Y., Du X., Lin Y., Li Z., Xia Q., Geng J., Dai H., Yu Y., Zhao X.D., Yuan Z., Yan J., Nie Q., Zhang X., Wang K., Chen F., Zhang Q., Zhu Y., Zheng S., Poss K.D., Tao S.C, & Meng X. (2020). An array of 60,000 antibodies for proteome-scale antibody generation and target discovery. Science Advances, 6(11), eaax2271.

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Mapping H3K36me3 Occupancy in Intestinal Epithelial Cells

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IECs were isolated from Setd2^f/f and Setd2^Vil−KO mice after 4 d of DSS treatment. The fragmented chromatin fragments were pre-cleared and then immunoprecipitated with Protein A + G Magnetic beads coupled with anti-H3K36me3 (Abcam, ab9050, 2–3μg/1 × 10⁶ cells) antibody and IgG antibody (CST, 2729S, 2–3μg/1 × 10⁶ cells). After reverse crosslinking, ChIP and input DNA fragments were end-repaired and A-tailed using the NEBNext End Repair/dA-Tailing Module (E7442, NEB) followed by adaptor ligation with the NEBNext Ultra Ligation Module (E7445, NEB). The DNA libraries were amplified for 15 cycles and sequenced using Illumina NextSeq 500 with single-end 1 × 75 as the sequencing mode. Raw reads were filtered to obtain high-quality clean reads by removing sequencing adapters, short reads (length<50 bp) and low-quality reads using Cutadapt (v1.9.1) and Trimmomatic (v0.35). Then FastQC is used to ensure high reads quality. The clean reads were mapped to the mouse genome (assembly GRCm38) using the Bowtie2 (v2.2.6) software. Peak detection was performed using the MACS (v2.1.1) peak finding algorithm with 0.01 set as the p-value cutoff. In total, 178919 and 217927 H3K36me3 peaks were identified in Setd2^Vil-KO and Setd2^f/f IECs over the input control, respectively. The heat maps and average profile for TSS were generated using ngsplot v2.61.

Liu M., Rao H., Liu J., Li X., Feng W., Gui L., Tang H., Xu J., Gao W.Q, & Li L. (2021). The histone methyltransferase SETD2 modulates oxidative stress to attenuate experimental colitis. Redox Biology, 43, 102004.

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