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.
Nebnext end repair da tailing module
Manufactured by New England Biolabs
Sourced in United States
The NEBNext End repair/dA-tailing Module is a kit that performs end repair and dA-tailing of DNA fragments in a single reaction. It converts the 5' and 3' ends of DNA into a form suitable for subsequent library construction.
Automatically generated - may contain errors
Lab products found in correlation
Neb blunt ta ligase master mix, by New England Biolabs (9 mentions)
Nebnext ultra ligation module, by New England Biolabs (8 mentions)
Ampure xp bead, by Beckman Coulter (6 mentions)
Nebnext ffpe dna repair mix, by New England Biolabs (4 mentions)
Hiseq 2000, by Illumina (3 mentions)
Maxima h minus reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Rnase cocktail enzyme mix, by Thermo Fisher Scientific (3 mentions)
Longamp taq master mix, by New England Biolabs (3 mentions)
Agencourt ampure xp magnetic beads, by Beckman Coulter (3 mentions)
Kapa hifi dna polymerase, by Roche (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Ligation sequencing kit primer mix, by Roche (2 mentions)
Nextseq 500, by Illumina (2 mentions)
Agencourt ampure xp, by Beckman Coulter (2 mentions)
Minion, by Oxford Nanopore (2 mentions)
Nanodrop one uv vis spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Agencourt ampure xp bead, by Beckman Coulter (2 mentions)
Nebnext ultra 2 ligation module, by New England Biolabs (2 mentions)
Nebuffer 1, by New England Biolabs (2 mentions)
Smrt cell 8m, by Pacific Biosciences (2 mentions)
32 protocols using nebnext end repair da tailing module
1
ChIP-Seq Protocol for Protein-DNA Interactions
2
Enriched DNA Sequencing with ONT
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We sequenced 1–1.5μg of the enriched DNA samples from the Xdrop workflow using the ONT platform, pooling two replicates when necessary. Amplified DNA was initially debranched using 15units of T7 endonuclease I in 30μl for 15min. Debranched DNA fragments were isolated by size selection using AmPure XP beads (Beckman Coulter, High Wycombe, United Kingdom) in the presence of 15% polyethylene glycol (Sigma–Aldrich, St Louis, MO, United States). The ONT sequencing library was generated using the Oxford Nanopore Ligation Sequencing Kit SQK-LSK109 (ONT, Oxford, United Kingdom) according to the manufacturer’s instructions with minor modifications. Briefly, DNA was end-repaired using the NEBNext FFPE DNA Repair Mix (New England Biolabs, Ipswich, MA, United States) at 20°C for 10min and subsequently end-prepped with the NEBNext End repair/dA-tailing Module (New England Biolabs) at 20°C for 20min. Sequencing adapters were ligated at room temperature for 10min. Finally, the 30–50 fmol library was loaded into a MinION R9.4.1 flowcell (ONT) and standard settings were applied for a run time of ~16h.
3
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.
4
Long-Read Sequencing of HSV-1 cDNA
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The 1D Strand switching cDNA by ligation protocol (Version: SSE_9011_v108_revS_18Oct2016) from the ONT was used for sequencing HSV-1 cDNAs on the MinION platform. The ONT Ligation Sequencing Kit 1D (SQK-LSK108) was applied for the library preparation using the recommended oligo(dT) primers, or custom-made random oligonucleotides, as well as the SuperScipt IV enzyme for the RTs. The cDNA samples were subjected to PCR reactions with KAPA HiFi DNA Polymerase (Kapa Biosystems) and Ligation Sequencing Kit Primer Mix (part of the 1D Kit). The NEBNext End repair/dA-tailing Module (New England Biolabs) was used for the end repair, whereas the NEB Blunt/TA Ligase Master Mix (New England Biolabs) was utilized for the adapter ligation. The enzymatic steps (e.g.: RT, PCR, and ligation) were carried out in a Veriti Cycler (Applied Biosystems) according to the 1D protocol (Moldován et al., 2018b (link); Tombácz et al., 2018b (link)). The Agencourt AMPureXP system (Beckman Coulter) was used for the purification of samples after each enzymatic reaction. The quantity of the libraries was checked using the Qubit Fluorometer 2.0 and the Qubit (ds)DNAHS Assay Kit (Life Technologies). The samples were run on R9.4 SpotON Flow Cells from ONT.
5
Nanopore Sequencing of Adult Female Genomes
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For ONT sequencing, we extracted HMW genomic DNA from 3 adult females. We prepared sequencing libraries using the Ligation Sequencing Kit (SQK-LSK109) (Oxford Nanopore Technologies, Oxford, UK), according to the manufacturer's protocols. Third-party reagents we used during library preparation included New England Biolabs (Ipswich, MA, USA) NEBNext End Repair/dA-Tailing Module (E7546), NEBNext FFPE DNA Repair Mix (M6630), and NEB Quick Ligation Module (E6056). We then sequenced the libraries, using ONT R.9.4.1 flowcells (FLO-PRO002), on an ONT PromethION (RRID:SCR_017987 ) sequencing platform. We then used ONT's Albacore basecalling software v.2.0.1 (RRID:SCR_015897 ) to basecall the raw fast5 data.
6
Bacterial mRNA Sequencing Library Prep
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Bacterial mRNA was isolated with the MICROBExpress™ kit (Invitrogen) by removing ribosomal RNA (rRNA) from total RNA according to the manufacturer’s instructions. The mRNA was fragmented and reverse transcribed with random hexamer primers (5’-d(NNNNNN)-3’ (N=G, A, T or C)) (Thermo Fisher Scientific) by using the Superscript™ Double-Stranded cDNA synthesis kit (Invitrogen). After repairing the cDNA ends using NEBNext End repair/dA-tailing module (New England Biolabs), sequencing adapter ligation was performed. Then, polymerase chain reaction (PCR) was carried out to generate the cDNA library. AMPure XP beads (Beckman Coulter Inc.) were utilized for purification after each enzymatic reaction. Following the construction of the cDNA library, preliminary quantification and the detection of the insert size were conducted by qubit 2.0 (Life Tech Invitrogen) and Agilent 2100, and the effective concentration of the library (> 2 nM) was accurately quantified by quantitative real-time PCR (qRT-PCR) to ensure the quality of the library. The cDNA library was established using NEBNext® UltraTM II RNA Library Prep Kit (New England Biolabs).
7
Mapping H3K36me3 Occupancy in Intestinal Epithelial Cells
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IECs were isolated from Setd2f/f and Setd2Vil−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 Setd2Vil-KO and Setd2f/f IECs over the input control, respectively. The heat maps and average profile for TSS were generated using ngsplot v2.61.
8
Direct cDNA Sequencing of BoHV-1 Infection
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Direct cDNA libraries were prepared from the mock and six BoHV-1 p.i samples in three replicates using the ONT’s Direct cDNA Sequencing Kit (SQK-DCS109) according to the manufacturer’s instructions. The first cDNA strand synthesis was performed using Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific) with SSP and VN primers (supplied in the kit) and 100 ng of poly(A) + RNA for each sample. This was followed by the removal of potential RNA contamination using RNase Cocktail Enzyme Mix (Thermo Fisher Scientific), and second strand synthesis using LongAmp Taq Master Mix (New England Biolabs). Double stranded cDNA ends were repaired using NEBNext End repair /dA-tailing Module (New England Biolabs). This was followed by ligation of sequencing adapter employing the NEB Blunt /TA Ligase Master Mix (New England Biolabs). Libraries were barcoded using Native Barcoding (12) Kit (ONT) according to the manufacturer’s instructions.
9
Oxford Nanopore Library Preparation Protocol
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The library preparation for Oxford Nanopore sequencing was performed using the manufacturer’s recommended protocol—1D Genomic DNA by ligation for the SQK-LSK108 kit (Oxford Nanopore Technologies, Oxford, UK). Briefly, 1 µg genomic DNA was end-prepped using the NEBNext End repair/dA-tailing Module (New England Biolabs, Inc., Ipswich, MA, USA) and the DNA was cleaned up using Agencourt AMPure XP (Beckman Coulter Inc., Brea, CA, USA). The DNA was then ligated to the adapter using NEB Blunt/TA Ligase Master Mix (NEB). The adapted library was purified using Agencourt AMPure XP (Beckman Coulter Inc.) and applied to a primed FLO-MIN106 R9.4 SpotON Flow Cell attached to MinION (Oxford Nanopore Technologies). The sequence reads were obtained with MinKNOW software using the 48-h protocol and live base-calling.
10
Illumina Sequencing Library Preparation
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About 0.5–5 ng of cDNA or ChIP‐precipitated DNA were used as starting material for the generation of sequencing libraries with the NEBNext Ultra II DNA library prep kit for Illumina (NEB). Alternatively, sequencing libraries were generated using the NEBNext End Repair/dATailingModule and NEBNext Ultra Ligation Module (NEB), followed by amplification with the KAPA Real‐Time Amplification kit (KAPA Biosystems). Cluster generation and sequencing were carried out using the Illumina HiSeq 2000/2500 system according to the manufacturer's guidelines. Dataset EV3 provides further information about all sequencing experiments of this study.
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