After germination of heterozygous rscΔ strains, the ploidy of each strain was monitored by flow cytometry (Fig. 1 b ). Genomic DNA from the sample that contained >95% diploid cells with the lowest number of generations was isolated. Genomic DNA from wild-type cells arrested in G1 with α-factor was used as the control sample. CGH was performed essentially as described previously (Dion and Brown, 2009 (link); Chambers et al., 2012 (link)). In brief, genomic DNA was amplified by using a WGA2 kit (Sigma) and treated with DNaseI (NEB) to fragment DNA to a mean 50-bp length. Then, DNA fragments were labeled with biotin-N6-ddATP (Enzo Life Sciences) by using terminal deoxynucleotidyl transferase (Fermentas), hybridized to an S. cerevisiae Tiling Array (Affymetrix), and visualized using streptavidin R-phycoerythrin conjugate (Invitrogen) and normal goat IgG (Sigma). Experiment signal intensities from each microarray were compared with the control sample using Tiling Analysis Software (Affymetrix) using quantile normalization, perfect match probes only, a bandwidth of 60, maximum gap of 80, and minimum run of 40. The CGH profiles were generated with IGB 6.3 (Affymetrix).
Wga2 kit
Manufactured by Merck Group
Sourced in Canada
The WGA2 kit is a laboratory product manufactured by Merck Group. The kit is designed for whole genome amplification, a process that generates multiple copies of the entire genome from a small amount of DNA. The core function of the WGA2 kit is to enable this amplification, providing researchers with a tool to produce sufficient DNA samples for various downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Mb400u, by Yasui Kikai (2 mentions)
Anti flag m2 antibody, by Merck Group (2 mentions)
Biotin n6 ddatp, by Enzo Life Sciences (1 mentions)
Streptavidin r phycoerythrin conjugate, by Thermo Fisher Scientific (1 mentions)
Normal goat igg, by Merck Group (1 mentions)
Tiling analysis software, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by New England Biolabs (1 mentions)
Terminal deoxynucleotidyl transferase, by Thermo Fisher Scientific (1 mentions)
Genechip s cerevisiae tiling 1.0r array, by Thermo Fisher Scientific (1 mentions)
Anti ha tag antibody chip grade, by Abcam (1 mentions)
Picogreen fluorescent dye, by Thermo Fisher Scientific (1 mentions)
Dneasy plant mini kit, by Qiagen (1 mentions)
Methylcollector ultra kit, by Active Motif (1 mentions)
Bioruptor sonicator, by Diagenode (1 mentions)
Anti flag m2 monoclonal antibody, by Merck Group (1 mentions)
Dynabeads sheep anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Pcr purification kit, by Qiagen (1 mentions)
Msei restriction enzyme, by New England Biolabs (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Dneasy kit, by Qiagen (1 mentions)
9 protocols using wga2 kit
1
Diploid Strain Identification by CGH
2
Yeast Oligonucleotide Tiling Microarray ChIP-chip
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S. cerevisiae oligonucleotide tiling microarrays were provided by Affymetrix (GeneChip S. cerevisiae Tiling 1.0R array). The high-density oligonucleotide arrays used allows the analysis of yeast chromosomes at a 300-bp resolution, each of the 300-bp region being covered by at least 60 probes. ChIP-chip of asynchronously growing cells was carried out as described [92 (link)] [93 (link)]. For immunoprecipitation with Rrm3-FLAG, cells growing in raffinose were shifted for four hours to galactose (2%) to overexpress YRA1. Overexpression after 4 hours was checked by Northern. Briefly, 1.5x107 cells were disrupted by multi-beads shocker (MB400U, Yasui Kikai, Japan), which maintained cells precisely at lower than 4°C during disruption. Anti-HA tag antibody (ChIP Grade, Abcam) and anti-FLAG antibody M2 (Sigma-Aldrich) were used for ChIP. ChIP DNA was purified and amplified by random priming using a WGA2 kit (Sigma- Aldrich) and following the manufacturer’s procedure. A total of 4 μg of amplified DNA was digested with DNaseI to a mean size of 100 bp and the purified DNA fragments were end-labelled with biotin-N6-ddATP23. The ChIP-chip data can be accessed at Gene Expression Omnibus (GSE68488; GSE68486).
3
Norway Spruce Genomic Resource Development
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Fresh needles were collected from 10 Norway spruce (Picea abies [L.] Karst.) grafted trees sampled in a 27-year old breeding orchard located north of Quebec City (Natural Resources Canada). All trees originated from central Europe, six of them being representative of distinct natural populations from Poland (3), Belorussia (1), and Latvia (2), and the four remaining ones being of unknown location. No permit was required to collect tissue in any location sampled in this study. DNA was isolated from needles using the Qiagen DNeasy Plant Mini Kit (Mississauga, ON, Canada) and quantified using the PicoGreen fluorescent dye (Invitrogen). Afterward, DNA samples were assembled in two pools of five individuals with equimolar concentrations [84 (link)]. In order to generate a reference sequence assembly with minimum genetic polymorphism, DNA was also extracted from a haploid megagametophyte, followed by whole-genome amplification using the WGA2 kit (Sigma-Aldrich, Oakville, ON, Canada).
4
Microarray-based DNA Methylation Profiling
5
Yeast Chromosome-Wide ChIP-Chip Analysis
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S. cerevisiae oligonucleotide tiling microarrays were provided by Affymetrix. The high-density oligonucleotide arrays used are able to analyze yeast chromosomes at a 300-bp resolution, each of the 300-bp region being covered by at least 60 probes. ChIP-chip of asynchronously growing cells was carried out as described (37 (link),38 (link)). Briefly, we disrupted 1.5×107 cells by multi-beads shocker (MB400U, Yasui Kikai, Japan), which was able to keep cells precisely at lower than 6°C during disruption by glass beads. Anti-FLAG monoclonal antibody M2 (Sigma-Aldrich) was used for ChIP. ChIP DNA was purified and amplified by random priming using a WGA2 kit (Sigma-Aldrich) and following the manufacturer's procedure. A total of 4 μg of amplified DNA was digested with DNase I to a mean size of 100 bp, purified, and the fragments were end-labeled with biotin-N6-ddATP23. ChIP-chip experiments shown with Thp1-FLAG and Sac3-FLAG are one per strain analyzed, which gave identical results. Rrm3-FLAG results shown are from two repetitions made for each strain analyzed, the data being normalized by the quantile method. The ChIP-chip data can be accessed at Gene Expression Omnibus (GSE56703, GSE56700).
6
Genome-wide Zfp148 Binding Profiling
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MEFs (1 × 108) transduced with AdZfp148FLAG were fixed in 1% formaldehyde. 200–500-bp DNA fragments were precipitated using the anti-FLAG M2 antibody (Sigma) conjugated to sheep anti-mouse IgG Dynabeads (Invitrogen), purified (QIAquick, Qiagen), amplified (WGA2 kit, Sigma), and hybridized to MM8_Deluxe_Promoter_HX1 chips at NimblGen (www.nimblegen.com ). The analysis was done on Zfp148gt/gt background to avoid competition with endogenous Zfp148 and Trp53−/− background to overcome senescence and obtain sufficient amounts of starting material. Gene ontology statistics was calculated using the DAVID software with “functional annotation clustering” setting for DAVID enrichment scores and “functional annotation chart” setting for ranking of functional terms37 (link),38 (link).
7
ChIP-seq protocol for Drosophila cells
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ChIP experiments from Drosophila tissue culture cells and third instar larvae were performed using a previously published method (59 (link)). For tissue culture cells, 1.5 × 108 S2 cells were harvested and processed, omitting the initial homogenization steps and instead directly resuspending the cells in cross-linking buffer. Precipitated DNA was used for quantitative real-time PCR (qRT-PCR) or whole genome amplification (WGA2 Kit, Sigma). The amplified DNA was purified using the Qiagen PCR purification kit and processed for microarray hybridization (2.1M Drosophila Whole-Genome Tiling Arrays, NimbleGen) according to the manufacturer's instructions (NimbleGen ChIP-chip User Manual).
8
Genome-Wide DNA Methylation Profiling
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Genomic DNA was extracted using the Qiagen DNeasy Kit, following the manufacturer’s protocol including the RNase A treatment. Genomic DNA was then digested into 200–1000 bp fragments by Mse I restriction enzyme (New England Biolabs). Immunoprecipitation (IP) of methylated genomic DNA was performed with a mouse monoclonal anti 5-methylcytidine antibody (Abcam, #10805). IP and input DNA were amplified with WGA2 Kit (Sigma-Aldrich). The amplified IP and input DNA were purified using the Qiagen QIAquick PCR Purification Kit (Catalog No. 28106) according to the manufacturer’s protocol. The IP and input gDNA were then labeled, hybridized, and scanned by the Keck Biotechnology Resource Laboratory using the Mouse DNA Methylation 3×720K CpG Island Plus RefSeq Promoter Array [Methylated DNA Immunoprecipitation (MeDIP)] (Nimble Gen). Data were analyzed by using Roche NimbleGen Software DEVA. Genes presenting a peak score greater than 3 were identified.
9
Chromatin Immunoprecipitation Protocols
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Protocols employed for ChIP, ChIP-chip DNase-chip have been previously described [25] (link), [29] (link). Primer sequences that were not previously published [25] (link) are available upon request. We employed a slightly modified ChIP protocol for p300 ChIP. Cells were dounced in 25 mM Hepes (pH 7.8), 1.5 mM MgCl2, 10 mM KCl, 0.1% NP-40, 1× complete protease inhibitor cocktail (Roche). Nuclei were isolated, resuspended in 0.1× SDS lysis buffer (Millipore) diluted in 1× ChIP dilution buffer (Millipore). Samples were sonicated in and subject to ChIP as previously described using ChIP assay kit (Millipore). For ChIP-chip, samples and inputs were amplified using WGA2 kit (Sigma), labeled and hybridized to custom-designed microarrays (Roche-Nimblegen). A previously described algorithm, ACME was employed for peak calling and identifying enriched regions in the ChIP-chip datasets [46] (link).
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