nebnext ultra end repair da tailing module, by New England Biolabs - Product details

1

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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2

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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3

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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4

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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5

Chromatin Immunoprecipitation and Sequencing

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ChIP and input DNA libraries were performed as previously
described^{63 (link)}. Briefly,
cells were cross-linked with 1% formaldehyde for 10min at room temperature and
formaldehyde was then inactivated by the addition of 125mM glycine for 5min.
Sonicated DNA fragments with 100–300bp were pre-cleared and
immunoprecipitated with Protein A+G Magnetic beads coupled with Anti-H3K27Ac
antibody (ab4729, Abcam) or Anti-YY1 antibody (#61779, active motif). 5μg
antibody per 1ml volume reaction was added for both antibodies. 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.
Analysis was carried out with ChIP-seq analysis pipeline on St. Jude
Cloud (https://platform.stjude.cloud/tools/chip-seq). Briefly, the reads
were aligned to the human genome (GRCh37-lite) with bwa^{55 (link)} (v0.7.12), then ambiguously-mapped and
duplicate reads were removed. Fragment length was estimated based on a
cross-correlation plot generated by SPP^{64 (link)} (v1.10.1). MACS^{65 (link)} (v2.1.1) was used to call the peaks. The results were
filtered against known false positive peaks using the ENCODE black
list^{66 (link)}.

Liu Y., Li C., Shen S., Chen X., Szlachta K., Edmonson M.N., Shao Y., Ma X., Hyle J., Wright S., Ju B., Rusch M.C., Liu Y., Li B., Macias M., Tian L., Easton J., Qian M., Yang J.J., Hu S., Look A.T, & Zhang J. (2020). Discovery of regulatory noncoding variants in individual cancer genomes by using cis-X. Nature genetics, 52(8), 811-818.

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6

Dual-end DNA Library Preparation for Nanopore Sequencing

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dsDNA was repaired and dA-tailed using 45 μl of dsDNA, 7 μl of Ultra II End-prep reaction buffer, 3 μl Ultra II End-prep enzyme mix (NEBNext Ultra End Repair/dA-Tailing Module, New England Biolabs) and 5 μl Nuclease-free water. The reaction mixture was incubated at 20°C for 5 min and 5 min at 65°C. AMPure bead purification was performed at 1:1 volumetric ratio of beads: DNA according to manufacturer's protocol. 15 μl of end-prepped DNA was mixed with 5 μl barcode adapter (1-96 barcoding kit, ONT) and 20 μl blunt/TA ligase master mix. The reaction mixture was incubated for 15 min at room temperature (RT). The adapter-ligated DNA was purified with AMPure bead at 0.4:1 volumetric ratio of bead: DNA and eluted in 26 μl NFW.
PCR-based barcoding was performed using 25 μl of adapter-ligated dsDNA, 2 μl of barcode and 50 μl of Long-Amp Taq 2X Master mix (New England Biolabs, Ipswich, MA). A reaction mixture of 100 μl was used for amplicon synthesis with the following conditions: denaturation at 95°C for 3 min; 17 cycles of denaturation at 95°C for 15 s, annealing at 62°C for 15 s and extension at 65°C for 90 s, and final extension of 65°C for 90 s and chilled at 4°C.

Butt S.L., Kariithi H.M., Volkening J.D., Taylor T.L., Leyson C., Pantin-Jackwood M., Suarez D.L., Stanton J.B, & Afonso C.L. (2022). Comparable outcomes from long and short read random sequencing of total RNA for detection of pathogens in chicken respiratory samples. Frontiers in Veterinary Science, 9, 1073919.

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7

cfDNA Ligation and TOP-PCR Amplification

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Prior to ligation, cfDNA fragments were end-repaired and 3′ A-tailed using NEBNext Ultra End Repair/dA-Tailing Module (NEB, E7442S/L). The ligation mixture (30 μL) contains end repaired/A-tailed cfDNA (normally ranging between 1 pg to a few ng), HA (at 50:1 ratio against cfDNA), 1X ligation buffer, and 1uL NEB Quick Ligation^TM Kit (NEB, M2200L). The reaction was incubated at 25 °C overnight in a thermocycler. The ligation mixture can be directly subjected to TOP-PCR amplification with or without purification. An at least 4X dilution is needed, if without purification.

Nai Y.S., Chen T.H., Huang Y.F., Midha M.K., Shiau H.C., Shen C.Y., Chen C.J., Yu A.L, & Chiu K.P. (2017). T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids. Scientific Reports, 7, 40767.

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8

Chromatin Immunoprecipitation and Sequencing

Cited 5 times

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ChIP and input DNA libraries were performed as previously
described^{63 (link)}. Briefly,
cells were cross-linked with 1% formaldehyde for 10min at room temperature and
formaldehyde was then inactivated by the addition of 125mM glycine for 5min.
Sonicated DNA fragments with 100–300bp were pre-cleared and
immunoprecipitated with Protein A+G Magnetic beads coupled with Anti-H3K27Ac
antibody (ab4729, Abcam) or Anti-YY1 antibody (#61779, active motif). 5μg
antibody per 1ml volume reaction was added for both antibodies. 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.
Analysis was carried out with ChIP-seq analysis pipeline on St. Jude
Cloud (https://platform.stjude.cloud/tools/chip-seq). Briefly, the reads
were aligned to the human genome (GRCh37-lite) with bwa^{55 (link)} (v0.7.12), then ambiguously-mapped and
duplicate reads were removed. Fragment length was estimated based on a
cross-correlation plot generated by SPP^{64 (link)} (v1.10.1). MACS^{65 (link)} (v2.1.1) was used to call the peaks. The results were
filtered against known false positive peaks using the ENCODE black
list^{66 (link)}.

Liu Y., Li C., Shen S., Chen X., Szlachta K., Edmonson M.N., Shao Y., Ma X., Hyle J., Wright S., Ju B., Rusch M.C., Liu Y., Li B., Macias M., Tian L., Easton J., Qian M., Yang J.J., Hu S., Look A.T, & Zhang J. (2020). Discovery of regulatory noncoding variants in individual cancer genomes by using cis-X. Nature genetics, 52(8), 811-818.

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9

Full-Length mRNA Sequencing via Oxford Nanopore

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The mixed RNA from each of the 18 samples was pooled in an equal quantity to obtain 1 ug RNA as required for the Oxford Nanopore library preparation. SuperScript IV First-Strand Synthesis System (Invitrogen) was used for full-length mRNA reverse transcription. According to the Oxford Nanopore recommended protocol, barcodes (Oxford Nanopore Technologies, ONT) were added to the RNA pool during cDNA amplification. The pooled cDNA was further end-repaired and dA-tailed using NEBNext Ultra End repair/dA-tailing module (NEB) and adaptor ligation using the T4 DNA ligase (NEB). Oxford Nanopore Technologies adapters were ligated to cDNA in a reaction containing adapter mix AMX1D (ONT) and Blunt/TA Ligase Master Mix (NEB). Libraries were purified by using Agencourt AMPure XP beads and eluted by Adapter Bead Binding buffer (ONT) and Elution Buffer (ONT). Libraries were then mixed with Fuel mix and Running buffer provided by ONT. Then, the final cDNA libraries were sequenced on one FLO-MIN109 flowcell and run on the PromethION platform.

Zhang Y., Yang X., Van de Peer Y., Chen J., Marchal K, & Shi T. (2022). Evolution of isoform-level gene expression patterns across tissues during lotus species divergence. The Plant journal : for cell and molecular biology, 112(3), 830-846.

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10

ChIP-seq Library Preparation Protocol

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280-1000 ng of ChIPed-DNA and input gDNA control were used for one library preparation. For end fill-in, NEBNext Ultra End Repair/dA-Tailing Module (New England Biolabs, Cat. E7442S) was used. For adapter ligation, NEBNext Ultra Ligation Module (New England Biolabs, Cat. E7445S) was used. 1X AMPure XP bead clean-up was performed to purify libraries after adapter ligation. For library amplification, KAPA HiFi HotStart ReadyMix (Kapa Biosystems, Cat. KK2601) was used. We used 6 cycles for input gDNA control and 12 cycles for ChIPed-DNA. After 0.8X AMPure XP bead clean-up, libraries were submitted to USC MGC.

Liang Y.C., Wu P., Lin G.W., Chen C.K., Yeh C.Y., Tsai S., Yan J., Jiang T.X., Lai Y.C., Huang D., Cai M., Choi R., Widelitz R.B., Lu W, & Chuong C.M. (2020). Folding Keratin Gene Clusters during Skin Regional Specification. Developmental cell, 53(5), 561-576.e9.

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Nebnext ultra end repair da tailing module

Lab products found in correlation

11 protocols using nebnext ultra end repair da tailing module

GUIDE-seq analysis of CRISPR off-target effects

ChIP-seq Library Preparation Protocol

ChIP-seq Analysis of AR and WT1 in Sertoli Cells

ChIP-Seq Library Preparation and Sequencing

Chromatin Immunoprecipitation and Sequencing

Dual-end DNA Library Preparation for Nanopore Sequencing

cfDNA Ligation and TOP-PCR Amplification

Chromatin Immunoprecipitation and Sequencing

Full-Length mRNA Sequencing via Oxford Nanopore

ChIP-seq Library Preparation Protocol

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