For the preparation of WGBS libraries, genomic DNA was first extracted from leaves and inflorescence tissue (for Ler samples) using the DNeasy Plant Mini Kit (Qiagen). 100 ng of DNA was then used for subsequent shearing using a Covaris S2 Focused Ultrasonicator. Libraries were then prepared using either the Ovation Ultralow Methyl-Seq kit (NuGen) in conjuction with the EpiTect Bisulfite Kit (Qiagen), or the Hyper Prep Kit (KAPA Biosystems) in conjuction with either the EZ DNA Methylation-Lightning Kit (Zymo) or the EpiTect Bisulfite Kit (Qiagen). Single-end 50 bp reads were then uniquely aligned to the TAIR10 genome using BS-Seeker258 (link). Methylation levels were then calculated for the CG, CHG, and CHH contexts. A filter was implemented to remove reads with three or more consecutively methylated cytosines in the CHH context, as previously described59 (link). Metaplots of BS-seq data were generated with custom Python and R scripts. For methylation calculations over individual chromosomes, each chromosome was split into 100 kb bins. Methylation values were then calculated from these bins.
Ovation ultralow methyl seq kit
Manufactured by Tecan
The Ovation Ultralow Methyl-seq kit is a laboratory equipment product designed for bisulfite sequencing to detect DNA methylation patterns. The kit enables low-input DNA preparation for genomic DNA methylation analysis.
Automatically generated - may contain errors
Lab products found in correlation
Epitect bisulfite kit, by Qiagen (6 mentions)
Epitect bisulfite conversion kit, by Qiagen (4 mentions)
Miseq 2 150, by Illumina (4 mentions)
Dneasy plant mini kit, by Qiagen (3 mentions)
Hiseq 4000, by Illumina (3 mentions)
Novaseq 6000, by Illumina (2 mentions)
Dneasy kit, by Qiagen (2 mentions)
E220 sonicator, by Covaris (2 mentions)
Truemethyl oxbs plugin, by Tecan (2 mentions)
S220 sonicator, by Covaris (2 mentions)
Allprep dna rna kit, by Qiagen (2 mentions)
Ez dna methylation lightning kit, by Zymo Research (1 mentions)
Hyper prep kit, by Roche (1 mentions)
S2 focused ultrasonicator, by Covaris (1 mentions)
Kapa dna hyper kit, by Roche (1 mentions)
Truseq dna adapters, by Illumina (1 mentions)
Hiseq 2000 platform, by Illumina (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Quick dna plant seed miniprep kit, by Zymo Research (1 mentions)
Ez dna methylation gold kit, by Zymo Research (1 mentions)
20 protocols using ovation ultralow methyl seq kit
1
WGBS Library Preparation and Analysis
2
Genome-wide Bisulfite Sequencing of Arabidopsis
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For WGBS, rosette leaf tissues from four- to five-week-old Col-0 and representative T2 MBD2–ZF108 transgenic lines that display the early flowering phenotype were collected and frozen in liquid nitrogen. For mature pollen WGBS, 1,000 μl of open flowers were collected, and the pollen pellets were immediately frozen after purification. DNA from leaf tissues and mature pollen were extracted using the DNeasy Plant Mini kit (Qiagen). A total of 500 ng of DNA from leaf tissues and 100 ng of DNA from mature pollen was sheared to ~300 bp using the Covaris S2 (Covaris). The libraries were then constructed using the Ovation Ultralow Methyl-seq kit (NuGEN), and bisulfite conversion was achieved using the Epitect Bisulfite Conversion kit (QIAGEN). Finally, the libraries were sequenced on Illumina NovaSeq 6000 or HiSeq 4000 instruments.
3
Genome-wide Bisulfite Sequencing for Plant DNA Methylation
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For T2 and T3 WGBS, DNA from inflorescences of adult plants was extracted following a CTAB-based method. Hundred nanograms of DNA were sheared to 200 bp with a Covaris S2 (Covaris) and used for library preparation using the Epitect Bisulfite Conversion kit (QIAGEN) and the Ovation Ultralow Methyl-seq kit (NuGEN) following the manufacturer’s instructions. For T4 and T5 WGBS, DNA from inflorescences of adult plants was extracted using the DNeasy Plant Mini Kit (QIAGEN). Two hundred fifty nanograms of DNA were sheared to 200 bp with a Covaris S2 (Covaris) and used for library preparation using the Epitect Bisulfite Conversion kit (QIAGEN) and the Kapa DNA hyper kit (Kapa Biosystems) with Illumina TruSeq DNA adapters following the manufacturer’s instructions.
4
Extracting and Shearing Plant DNA
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DNA from leaves of one adult plant grown on soil was extracted following a CTAB-based method. 100ng of DNA was sheared to 200bp with a Covaris S2 (Covaris) and used for library preparation using the Epitect Bisulfite Conversion kit (QIAGEN) and the Ovation Ultralow Methyl-seq kit (NuGEN) following the manufacturer’s instructions.
5
Zinc Finger Protein 108 Targets Multiple Loci in Arabidopsis Genome
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Example 12
This Example illustrates additional regions of the Arabidopsis genome targeted by the ZF108 artificial zinc finger.
Materials and Methods
DNA from leaves of different lines expressing ZF108 fused to various factors was extracted by a CTAB-method. Libraries for whole genome bisulfite sequencing were prepared using the Ovation Ultralow methyl-seq kit (Nugen) and sequenced using the HiSeq 2000 platform following manufacturer instructions (Illumina) at a length of 50 bp. Bisulfite-Seq (BS-Seq) reads were aligned to the TAIR10 version of the Arabidopsis thaliana reference genome using BS-seeker. For BS-Seq up to 2 mismatches were allowed and only uniquely mapped reads were used.
Results
The results demonstrate DNA methylation targeting at a second location in the Arabidopsis genome with two sequences very similar to the ZF108 target locus. Both Col0 and fwa-4 plants have very little pre-existing methylation at this site, but the ZF108 fusion protein containing plants contain methylation in all three sequence contexts (
6
Bisulfite Sequencing of Paramecium Genome
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For bisulfite sequencing, total genomic DNA extracted from different time points were sent to the Lausanne Genomic Technologies Facility at the University of Lausanne. Library preparation with bisulfite conversion was performed with the Ovation Ultralow Methyl-Seq kit (Nugen) after spiking in Phi-X lambda DNA to ~0.5% total mass. Paired-end sequencing was performed on Illumina HiSeq 2500. Sequencing reads in the form of fastq files were processed following the suggested protocol on Bismark website using version 0.18.1 (https://rawgit.com/FelixKrueger/Bismark/master/Docs/Bismark_User_Guide.html ). First, reads were quality trimmed and filtered using Trim Galore[34 ], [35 ]. Filtered reads were then mapped to the Paramecium macronuclear genome (ptetraurelia_mac_51.fa) with options–n 1 and–X 1000. Methylation calls were then extracted for all C’s with bismark_methylation_extractor command. Bismark Cytosine reports were combined, processed and analyzed in R.
Human methylation dataset (SRR34552)[36 ] was used as a positive control for the analysis workflow. Reads were mapped to human GRC37.
Human methylation dataset (SRR34552)[36 ] was used as a positive control for the analysis workflow. Reads were mapped to human GRC37.
7
Whole Genome Bisulfite Sequencing of Arabidopsis
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Genomic DNA was extracted from aerial parts of 3-week-old seedlings using Quick-DNA Plant/Seed Miniprep Kit (Zymo research, D6020) according to the manufacturer’s protocol. Whole genome bisulfite sequencing (WGBS) library was prepared from 50 ng genomic DNA using NuGen Ovation Ultralow Methyl-Seq kit. Bisulfite treatment was carried out by Qiagen Epitect bisulfite kit. WGBS libraries were sequenced on an Illumina HiSeq 4000 machine. The raw reads (single end) were trimmed using Trimmomatic in order to remove adapter sequences [35 (link)]. The remaining sequences were aligned against the A. thaliana genome TAIR10 version using Bismark [43 (link)]. Duplicated reads were collapsed into one read. For metaplot and boxplot visualization, we used ViewBS [44 (link)]. Boxplots were realized using boxplot function from R. DMRs (differentially methylated regions) were defined comparing methylation in wildtype with the main-2 mutant analyzed using the R package “DMRcaller” [45 (link)]. We used “noise filter” method to compute CpG, CpHpG and CpHpH DMRs. We selected bins where the p-value was less than 0.01, the difference in methylation level was at least 40% in the CG context, 20% in the CHG context or 10% in the CHH context, with at least four cytosines; each cytosine had on average at least four reads.
8
Whole Genome Bisulfite Sequencing and Analysis
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CTAB-based method was used to extract DNA. Approximately 75 to 150 ng of DNA was used to synthesize whole genome bisulfite sequencing libraries using the Ovation Ultralow Methyl-seq kit (NuGEN). Raw sequencing reads were aligned to the TAIR10 genome using Bismark [38 (link)]. Methylation ratios are calculated by #C/(#C+#T) for all CG, CHG, and CHH sites by a custom bash script. This was used to generate bigwig files for tracks to view on Integrative Genomics Viewer (IGV). Cytosines with more than 5 read coverage and zero methylation were represented by a negative black bar, while cytosines with less than 5 read coverage are represented by no bars. Reads with three consecutive methylated CHH sites were discarded since they are likely to be unconverted reads as described before [39 (link)]. Whole genome metaplots were generated using the program ViewBS and following instructions of authors [40 (link)]. The same program was also used to calculate the genome wide methylation.
9
Comprehensive Workflow for Characterizing Pluripotent Stem Cells
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All husbandry and experiments involving mice were authorized by a UK Home Office Project License 80/2637 and carried out in a Home Office-designated facility.
ESCs were maintained on gelatin-coated dishes using culture medium as specified inFigure 1 A, for at least five passages prior to experimental analysis. ESCs were maintained in a humidified 37°C chamber supplemented with 5% CO2 and passaged every 2–3 days with TrpLE. RNA-seq was performed on independent replicate samples using the TruSeq RNA Library Preparation v. 2.0 Kit (Illumina). Whole-genome bisulfite sequencing was carried out using the EZ DNA Methylation Gold Kit (Zymo Research) and Ovation Ultralow Methyl-seq Kit (NuGEN). Libraries were sequenced on a HiSeq 1500 or 2500. Differentiation assays were performed as described previously (Ying et al., 2003 (link), Hayashi et al., 2011 (link)). CRISPR-mediated knockout of Klf4 was achieved using dual gRNAs and the Cas9 nickase to target a critical portion of exon 3. A complete description of all methods and bioinformatics analysis is listed in the Supplemental Experimental Procedures .
ESCs were maintained on gelatin-coated dishes using culture medium as specified in
10
Profiling Epigenetic Alterations in CD34+ Cells
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Libraries for whole-genome bisulfite sequencing were generated from 5 ng of purified DNA from paired senescent and active CD34+ cells from 3 healthy human donors (33, 45, and 53 years of age) using the NuGen Ovation Ultralow MethylSeq Kit following the manufacturer’s protocol, for a total of 6 individual libraries. Samples were not pooled prior to library generation. Reads were aligned using Biscuit and Metilene was used for calling of differentially methylated regions. Motif analysis of DMRs was conducted using the PWMEnrich package with Hocomoco and Factorbook motif databases provided in the motifbreakR package [30 ].
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