Genomic DNA was isolated with the Qiagen Blood and Tissue Kit according to the manufacturer’s instructions. 0.5% (w/w) of unmethylated lambda phage DNA (Promega) was added to the sample genomic DNA for the purpose of an unmethylated control to measure the bisulfite non-conversion frequency in each sample. Genomic DNA was fragmented with either either a Covaris S2 sonicator or a Covaris M220 sonicator to a mean length of 200 bp, then end-repaired, A-tailed, ligated to methylated Nextflex Bisulfite-Seq barcodes (Perkin Elmer) using the NxSeq AmpFREE low DNA library kit (Gene Target Solutions) and subjected to PCR amplification with KAPA HiFi Uracil+ DNA polymerase (KAPA Biosystems)56 (link). Sequencing was performed single-end on a HiSeq 1500, NextSeq 500, or paired-end on a NovaSeq 6000 (Illumina).
Unmethylated lambda phage dna
Manufactured by Promega
Sourced in United States
Unmethylated lambda phage DNA is a laboratory reagent that consists of purified DNA from the lambda bacteriophage. It has not been methylated and retains the natural DNA structure of the phage. This DNA can be used as a control or reference material in various molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Nextflex bisulfite seq barcodes, by PerkinElmer (5 mentions)
Novaseq 6000, by Illumina (3 mentions)
Kapa library quantification kit, by Merck Group (3 mentions)
Hiseq x platform, by Illumina (3 mentions)
Ez dna methylation gold kit, by Zymo Research (3 mentions)
Hiseq 1500, by Illumina (3 mentions)
Blood and tissue kit, by Qiagen (2 mentions)
Kapa hifi uracil dna polymerase, by Roche (2 mentions)
M220 sonicator, by Covaris (2 mentions)
S2 sonicator, by Covaris (2 mentions)
Kapa hifi hotstart uracil dna polymerase, by Roche (2 mentions)
Kapa library quantification kit for platform, by Illumina (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
Kapa library amplification kit, by Roche (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Real time analysis v2, by Illumina (1 mentions)
Dsdna hs, by Agilent Technologies (1 mentions)
Bcl2fastq v2, by Illumina (1 mentions)
Accel ngs methyl seq dna library kit, by Integrated DNA Technologies (1 mentions)
Methyl seq combinatorial dual indexing kit, by Integrated DNA Technologies (1 mentions)
17 protocols using unmethylated lambda phage dna
1
Bisulfite Sequencing Library Preparation
2
Whole Genome Bisulfite Sequencing Protocol
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DNA was isolated from cells using the Qiagen AllPrep DNA/RNA Minikit and quantified using a Qubit fluorometer. Prior to library preparation, sample DNA was spiked with unmethylated lambda phage DNA (Promega) at a concentration of 5 ng lambda DNA/1 μg sample DNA. DNA was fragmented to approximately 350 bp using a Covaris M220 Sonicator, and bisulfite-converted using the Zymo EZ DNA Methylation-Gold Kit according to manufacturer’s instructions (Zymo Research). Bisulfite sequencing libraries were prepared using the Accel-NGS Methyl-Seq DNA Library Kit and Methyl-Seq Combinatorial Dual Indexing Kit (Swift Biosciences). Completed libraries were quantified and QC’ed using Qubit dsDNA HS and Agilent 4200 TapeStation HS DNA1000 assays, respectively.
Sequencing libraries were divided into three pools of six libraries, and WGBS was performed on each pool across three flow cell lanes on an Illumina HiSeq 4000 instrument in 2 × 150PE format using HiSeq 4000 reagents. A PhiX control DNA library was spiked into each lane at 10% of the total to account for the unbalanced base composition inherent in Methyl-Seq libraries. Base calling was done by Illumina Real Time Analysis (RTA) v2.7.7 and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v2.19.1.
Sequencing libraries were divided into three pools of six libraries, and WGBS was performed on each pool across three flow cell lanes on an Illumina HiSeq 4000 instrument in 2 × 150PE format using HiSeq 4000 reagents. A PhiX control DNA library was spiked into each lane at 10% of the total to account for the unbalanced base composition inherent in Methyl-Seq libraries. Base calling was done by Illumina Real Time Analysis (RTA) v2.7.7 and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v2.19.1.
3
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).
4
Zebrafish Whole Genome Bisulfite Sequencing
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WGBS libraries were prepared from 500 ng of zebrafish gDNA spiked with 0.025ng of unmethylated lambda phage DNA (Promega, Madison, WI, USA). The DNA was sonicated to an average insert size of 300 bp followed by end repair and overnight ligation of adapters using NEXTFLEX Bisulfite-Seq barcodes (PerkinElmer, Waltham, MA, USA). DNA was bisulphite-converted using EZ DNA Methylation Gold Kit (Zymo Research, Irvine, CA, USA) according to manufacturer's instructions. Library amplification was performed with KAPA HiFi HotStart Uracil+ DNA polymerase (Kapa Biosystems, Woburn, MA), using 8 cycles of amplification. Library concentration was quantified through qPCR using KAPA Library Quantification Kit (Sigma-Aldrich, St. Louis, MO, USA) according to manufacturer's instructions. The combined libraries with 15% PhiX spike-in were sequenced on the Illumina HiSeqX platform (150 bp paired-end sequencing, high output mode).
5
WGBS Library Preparation and Sequencing
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WGBS was performed using the post-bisulfite adaptor-tagging (PBAT) method66 (link),67 (link). Briefly, genomic DNA was purified from hTSLprimed cells with phenol/chloroform extraction and ethanol precipitation. Genomic DNA spiked with 0.5% (w/w) unmethylated lambda phage DNA (Promega) was used for library preparation according to the PBAT protocol. These DNA samples were treated with sodium bisulfite using EZ DNA Methylation-Gold Kit (Zymo Research). Concentrations of the PBAT products were quantified using the KAPA Library Quantification Kit for Illumina platforms (Kapa Biosystems). PBAT libraries were sequenced on the Illumina HiSeq 2500 (Illumina) with 101 bp single-end reads. The reads were aligned to the reference genome (UCSC hg38) using Bismark (v0.19.1)68 (link). The methylation level of each CpG site was calculated using the Bismark methylation extractor. We confirmed bisulfite conversion rates of >99% for all samples. For each CpG site, reads from both strands were combined to calculate the methylation level. Methylation levels of CpGs covered with ≥5 reads were analyzed.
6
Bisulfite Sequencing Library Preparation
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Genomic DNA was isolated with the Qiagen Blood and Tissue Kit according to the manufacturer’s instructions. 0.5% (w/w) of unmethylated lambda phage DNA (Promega) was added to the sample genomic DNA for the purpose of an unmethylated control to measure the bisulfite non-conversion frequency in each sample. Genomic DNA was fragmented with either either a Covaris S2 sonicator or a Covaris M220 sonicator to a mean length of 200 bp, then end-repaired, A-tailed, ligated to methylated Nextflex Bisulfite-Seq barcodes (Perkin Elmer) using the NxSeq AmpFREE low DNA library kit (Gene Target Solutions) and subjected to PCR amplification with KAPA HiFi Uracil+ DNA polymerase (KAPA Biosystems)56 (link). Sequencing was performed single-end on a HiSeq 1500, NextSeq 500, or paired-end on a NovaSeq 6000 (Illumina).
7
Plasma cfDNA Extraction and Isolation
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Plasma samples were isolated from whole blood collected in Cell-Free DNA BCT® (Streck, La Vista, NE) or EDTA (BD) tubes by centrifugation at 1600g for 10 min at 4°C, aliquoted and stored immediately at −80°C. The aliquoted plasma was thawed at room temperature and centrifuged at 16000g for 5 min at 4 °C to remove residual debris. cfDNA was extracted from 1 mL of plasma by QIAsymphony circulating DNA kit (QIAGEN). The plasma samples were spiked with 0.142 ng/mL unmethylated lambda phage DNA (Promega), which was fragmented to 160 bp, to measure the efficiencies of cfDNA extraction and bisulfite conversion for genome-wide methylation sequencing. The isolated cfDNA were eluted into 60 μL elution buffer and frozen at −20 °C until ready for further use.
8
Zebrafish DNA methylation analysis
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RRBS libraries were prepared from 500 ng of zebrafish gDNA spiked with 0.025 ng of unmethylated lambda phage DNA (Promega, Madison, WI, USA). The DNA was digested for 2 h with 10 U BccI and 10U SspI (New England BioLabs, Ipswich, MA, USA), with the exception of uhrf1 cKO libraries which were digested with 20 U MspI. 5′ overhangs of the digested DNA were filled-in and A-tailed using Klenow fragment exo- (New England BioLabs, Ipswich, MA, USA), followed by an overnight ligation of NEXTFLEX Bisulfite-Seq barcodes (PerkinElmer, Waltham, MA, USA). DNA was bisulphite-converted using EZ DNA Methylation Gold Kit (Zymo Research,Irvine,CA,USA), according to manufacturer's instructions. Library amplification was performed with KAPA HiFi HotStart Uracil+ DNA polymerase (Kapa Biosystems, Woburn, MA, USA), using 13 cycles of amplification. Library concentration was quantified by qPCR using KAPA Library Quantification Kit (Sigma-Aldrich, St. Louis, MO, USA) according to manufacturer's instructions. The combined libraries with 15% PhiX spike-in were sequenced on the Illumina HiSeqX platform (150 bp paired-end sequencing, high output mode).
9
WGBS of Pooled Fetal Germ Cells
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WGBS libraries were constructed using the post-bisulfite adaptor tagging method47 (link). Pooled FGOs (150–200 cells per sample) were spiked with 1% unmethylated lambda phage DNA (Promega). Libraries were amplified using the KAPA library amplification kit (KK2620, KAPA) for four cycles. The resulting libraries were sequenced on an Illumina NovaSeq 6000 (108 bp, single-end).
10
Oocyte Methylome Analysis by RRBS and PABT
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For RBBS, the oocytes were collected in pools of between 250 and 500 oocytes depending on the size of the oocyte and were carried in replicate for each condition 20% O2 and 5% O2 at two different oocyte size (class I and IV). Libraries for RRBS were prepared as described previously by Smallwood et al. [3 (link)]. Genomic DNA from approximately 300 cultured oocytes was extracted using QIAamp DNA Micro Kit (Qiagen) and was spiked with 10 fg of unmethylated lambda phage DNA (Promega) followed by MspI digestion (Fermentas), and the libraries were generated according to Smallwood et al. [3 (link)]. Libraries runs, in duplicates, were single-ended, and 100 base pairs (bps) in length were sequenced on Illumina HiSeq 2500 platform.
Post Bisulfite Amplification Treatment (PABT) libraries were generated as described previously by Peat et al. [41 (link)] for whole genome sequencing. 70–100 cultured oocytes under different treatments (Tranylcypromine, LiCl, Tranycyltramine + LiCl, and control also called 20% O2 CON) with a size of 55–65 μm were used to generate the libraries in triplicate. Libraries runs were single-ended, and 100 bps in length were sequenced on Illumina HiSeq 2500 platform.
Post Bisulfite Amplification Treatment (PABT) libraries were generated as described previously by Peat et al. [41 (link)] for whole genome sequencing. 70–100 cultured oocytes under different treatments (Tranylcypromine, LiCl, Tranycyltramine + LiCl, and control also called 20% O2 CON) with a size of 55–65 μm were used to generate the libraries in triplicate. Libraries runs were single-ended, and 100 bps in length were sequenced on Illumina HiSeq 2500 platform.
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