The entire amount of extracted plasma cfDNA (capped at 15 ng if exceeding what is needed) for each sample will be used to generate WMS libraries with the NEBNext Enzymatic Methyl-seq Kit (New England Biolabs) according to manufacturer instructions, with 1 modification: 100 ng of carrier RNA (TIANGEN) will be added before denaturation by sodium hydroxide. The generated libraries will be amplified with 9 polymerase chain reaction cycles, followed by quantification using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). After that, the constructed libraries will be subjected to sequencing on a NovaSeq 6000 system (Illumina) with a paired-end read length of 100 bp.
Nebnext enzymatic methyl seq kit
Manufactured by New England Biolabs
Sourced in United States
The NEBNext Enzymatic Methyl-seq Kit is a complete solution for genome-wide DNA methylation analysis. It enables bisulfite-free detection of 5-methylcytosine and 5-hydroxymethylcytosine through enzymatic treatment of DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Novaseq 6000, by Illumina (8 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (6 mentions)
Novaseq 6000 system, by Illumina (4 mentions)
Novaseq s4 lane, by Illumina (2 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (2 mentions)
Labchip gx, by PerkinElmer (2 mentions)
Nextseq 550, by Illumina (2 mentions)
Quick dna microprep kit, by Zymo Research (2 mentions)
S220 focused ultrasonicator, by Covaris (2 mentions)
Nextseq 2000, by Illumina (2 mentions)
Dneasy blood and tissue kit, by Qiagen (2 mentions)
Qubit dna assay kit in qubit 3.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Sequencing platform, by Illumina (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Unmethylated lambda phage dna, by Promega (1 mentions)
Nextflex bisulfite seq barcodes, by PerkinElmer (1 mentions)
Kapa library quantification kit, by Merck Group (1 mentions)
Puc19 plasmid, by New England Biolabs (1 mentions)
Hiseq x platform, by Illumina (1 mentions)
Tapestation 4200, by Agilent Technologies (1 mentions)
31 protocols using nebnext enzymatic methyl seq kit
1
Enzymatic Methyl-seq Library Prep for cfDNA
2
EM-Seq Analysis of DNA Methylation
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Single biological replicates of the fifth true leaf of 3-week-old WT and drdd individuals were collected. DNA was extracted using a CTAB protocol and enzymatic methyl sequencing (EM-Seq) was carried out using 100 ng of input DNA. Input DNA was first sheared in 1× TE buffer using a Covaris S220 to an average fragment size of 200 bp (175W, 10% duty factor, 200 cycles/burst, 180 s). After shearing, a 2.5× bead cleanup was performed with a final elution in water. EM-seq was conducted using the NEBNext Enzymatic Methyl-Seq Kit according to the manufacturer’s directions; including the provided spike-in controls (methylated and unmethylated DNA). Six cycles of PCR amplification were performed. Samples were equally multiplexed across three lanes and sequenced using an Illumina HiSeq2500 standard run with 100 bp paired-end reads at the Whitehead Institute Genome Technology Core.
3
DNA Methylation Analysis of Diverse Samples
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Cells (about 1 × 109 cells) were Collected and used the QIAamp DNA Mini Kit (Qiagen, Germany) to extracted DNA according to the manufacturer’s instructions. Then DNA concentration was measured using Qubit® DNA Assay Kit in Qubit 3.0 Fluorometer (Life Technologies, CA, United States) and DNA quality was assessed by 1% agarose gel electrophoresis. Methylation libraries were constructed by using the NEBNext® Enzymatic Methyl-seq Kit (NEB) with 200 ng DNA as input amounts. And referred to the manufacturer’s protocol, we prepped libraries with fragments size of ∼400 bp for each sample (no biological replicates, a total of 3 libraries). High-throughput sequencing was performed using the Illumina sequencing platform, and the sequencing read length was 150 bp paired-end reads.
We got 3.3G of raw data in total. And bismark (Krueger and Andrews, 2011 (link)) was used to identify and extract methylation sites. The R package DSS (Wu et al., 2015 (link)) was used to identify differentially methylated regions of different components, using a threshold of P-value < 0.005.
We got 3.3G of raw data in total. And bismark (Krueger and Andrews, 2011 (link)) was used to identify and extract methylation sites. The R package DSS (Wu et al., 2015 (link)) was used to identify differentially methylated regions of different components, using a threshold of P-value < 0.005.
4
Whole-Genome Bisulfite Sequencing Protocol
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WGBS was performed by the Center d’expertize et de services Génome Québec at McGill University. Genomic DNA was quantified using the Quant-iT™ PicoGreen® dsDNA Assay Kit (Life Technologies). 2 × 151 bp paired-end libraries were generated using the NEBNext® Enzymatic Methyl-seq Kit (New England BioLabs, NEB). Adapters were purchased from NEB. Libraries were quantified using the Kapa Illumina GA with Revised Primers-SYBR Fast Universal kit (Kapa Biosystems) and average size fragment was determined using a LabChip GX (PerkinElmer) instrument. The libraries were normalized and pooled and then denatured in 0.05 N NaOH and neutralized using HT1 buffer. The pool was loaded at 225 pM on an Illumina NovaSeq S4 lane using Xp protocol as per the manufacturer’s recommendations. The run was performed for 2 × 100 cycles (paired-end mode). A phiX library was used as a control and mixed with libraries at 5% level. Base calling was performed with RTA v3.4.4. Program bcl2fastq2 v2.20 was then used to demultiplex samples and generate FastQ reads.
5
Enzymatic Methyl-seq Library Prep
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EM-seq library construction was performed using the NEBNext Enzymatic Methyl-seq Kit (New England BioLabs, Ipswich, MA, USA) according to manufacturer's instructions with minor modifications. 0.02 ng of unmethylated lambda phage DNA (Promega, Madison, WI, USA) and 0.0001 ng of pUC19 plasmid methylated at 100% of CpG sites (New England BioLabs, Ipswich, MA, USA) were used as spike-in controls to determine the efficiency of APOBEC deamination and TET2 oxidation, respectively. Briefly, 200 ng of zebrafish gDNA was sonicated to an average insert size of 300 bp. Input DNA concentration was selected according to the optimal input amount as recommended by the manufacturer. Sonicated DNA was end-repaired followed by ligation of adapters to DNA overnight using NEXTFLEX Bisulfite-Seq barcodes (PerkinElmer, Waltham, MA, USA). DNA was treated with TET2 for 1 h. Following TET2 oxidation, DNA was denatured with 0.1 M NaOH then treated with APOBEC for three hours. DNA was then PCR-amplified (8 cycles). Library concentration was quantified by qPCR using KAPA Library Quantification Kit (Sigma-Aldrich, St. Louis, MO, USA). 150pmol of the combined libraries with 15% PhiX spike-in was sequenced on the Illumina HiSeqX platform (150 bp paired-end sequencing, high output mode).
6
Targeted DNA Methylation Analysis by NGS
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Purified gDNA was sonicated into 200–300 bp fragments using Covaris S2 (Covaris Inc. Woburn, MA, USA). We used 200 ng of gDNA to construct sequencing libraries, which were sequenced using the Illumina NovaSeq 6000. The sequencing libraries were prepared using a NEBNext Enzymatic Methyl-seq Kit (New England Biolabs, Ipswich. MA, USA) and subjected to target enrichment with a Twist methylome panel (covering 85 Mbp of the human genome) and Twist Targeted Methylation Workflow (Twist Biosciences, San Francisco, CA, USA) according to the manufacturer's instructions.
Sequencing libraries were diluted to a concentration of 2 nM based on DNA concentration and average fragment size and then pooled in equal volumes. The pooled libraries were denatured and then subjected to cluster amplification according to the manufacturer's instructions (Illumina, San Diego, CA, USA). Flow cells were sequenced in 150 bp paired-end mode using NovaSeq 6000 or NextSeq 550 (Illumina, San Diego, CA, USA) and then analyzed using RTA v.1.12.4.2 or later.
Sequencing libraries were diluted to a concentration of 2 nM based on DNA concentration and average fragment size and then pooled in equal volumes. The pooled libraries were denatured and then subjected to cluster amplification according to the manufacturer's instructions (Illumina, San Diego, CA, USA). Flow cells were sequenced in 150 bp paired-end mode using NovaSeq 6000 or NextSeq 550 (Illumina, San Diego, CA, USA) and then analyzed using RTA v.1.12.4.2 or later.
7
Enzymatic Methyl-seq Library Prep
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Leaf tissue from BZR1-YFP/B73xMo17 F1s was harvested (n=6 plants, 3 replicates) and treated the same way (including BL treatment) as described for ChIP but without crosslinking. Tissues were homogenized in liquid nitrogen, and DNA was isolated with the DNeasy Plant Mini Kit (Qiagen). Libraries were prepared using the NEBNext Enzymatic Methyl-seq Kit (NEB) following the protocol for large DNA inserts. Therefore, 200ng genomic DNA was combined with 0.002 ng CpG methylated pUC19 DNA and 0.04 ng unmethylated lambda DNA. Fragmentation was done by using the Diagenode Bioruptor NGS in three rounds, 30s on, 90s off. Agilent Technologies 4200 Tape Station was used to determine the size distribution and concentration of the libraries.
8
Genomic DNA Isolation and Bisulfite Sequencing
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Genomic DNA was isolated from sorted cells using a Quick-DNA Microprep Kit (Zymo) and sonicated to generate fragments of approximately 350 to 400 base pairs using S220 Focused Ultrasonicator (Covaris). Unmethylated cytosines were converted to uracils, and sequencing libraries were created using the NEBnext Enzymatic Methyl Seq Kit (New England Biolabs) according to the manufacturer’s instructions. DNA libraries were sequenced using an Illumina NovaSeq 6000 system following the manufacturer’s protocols.
9
Bisulfite Sequencing of Mouse Genomic DNA
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In this, 200 ng of purified genomic DNA was used to create bisulfite sequencing libraries via the large DNA insert approach using the NEBNext® Enzymatic Methyl-seq Kit (New England Biolabs). In short, purified genomic DNA was fragmented, adenylated, and ligated to EM-seq adapters. Bisulfite conversion was performed according to the manufacturer’s instructions. Internal unmethylated lambda DNA and CpG methylated pUC19 DNA controls were included with each library. Libraries were sequenced with a NovaSeq 6000 instrument (Illumina) to generate a minimum of 300 million paired-end 151 base reads per library.
Raw fastq files were quality trimmed using TrimGalore (version 0.6.7). Reads were aligned to the mouse genome (mm10) using Bismark85 (link) (version 0.23.0). Mapped BAM files were sorted by query name, deduplicated using Picard (version 2.27.3) and finally sorted coordinates using Samtools86 (link) (version 1.17). Integrative Genomics Viewer74 (link) (version 2.5.3) was used for visualization.
Raw fastq files were quality trimmed using TrimGalore (version 0.6.7). Reads were aligned to the mouse genome (mm10) using Bismark85 (link) (version 0.23.0). Mapped BAM files were sorted by query name, deduplicated using Picard (version 2.27.3) and finally sorted coordinates using Samtools86 (link) (version 1.17). Integrative Genomics Viewer74 (link) (version 2.5.3) was used for visualization.
10
Simultaneous RNA-seq and EM-seq of Mouse Cortex
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