The nascent chromatin capture assay was performed as described previously (48 (link)) with slight modifications. Cells were treated with a mixture of biotin-16-dUTP and biotin-16-dCTP (0.5 μM each, Jena Bioscience) in hypotonic buffer (50 mM KCl, 10 mM HEPES) for 5 min and followed by another 5 min dUTP/dCTP treatment in regular DMEM medium (For etoposide treated samples, etoposide was added 20 min prior dUTP/dCTP treatment and kept until cells were fixed). Cells were then fixed by 0.2% PFA for 5 min at room temperature and quenched by co-incubation with 200 mM glycine for 1 min. Cells were resuspended in TNT-300 buffer (25 mM Tris–HCl pH 7.4, 300 mM NaCl, 1% Triton-X100) together with protease and phosphatase inhibitors (Sigma, 1:1000) and sonified (30% amplitude, 45 s on/15 s off for 10 min) before incubated with 10 μl of Streptavidin Magnetic Beads (50% slurry, NEB) at room temperature 45 min on a rotating wheel. Beads were collected by centrifugation and washed three times with the TNT-300 buffer. Samples were denatured by incubation with the 4× Laemmli loading buffer at 95°C for 10 min and analyzed by immunoblotting.
Biotin 16 dutp
Manufactured by Jena Biosciences
Sourced in Germany, Japan
Biotin-16-dUTP is a nucleotide analog that contains a biotin molecule covalently attached to the 16th carbon of the pyrimidine ring of dUTP. It is commonly used in various molecular biology and biotechnology applications, such as DNA labeling and detection.
Automatically generated - may contain errors
Lab products found in correlation
Digoxigenin 11 dutp, by Jena Biosciences (4 mentions)
Qiaquick pcr purification kit, by Qiagen (3 mentions)
Ex taq polymerase, by Takara Bio (2 mentions)
Protease and phosphatase inhibitor, by Merck Group (1 mentions)
Streptavidin magnetic beads, by New England Biolabs (1 mentions)
Biotin 16 dctp, by Jena Biosciences (1 mentions)
Terminal transferase, by Merck Group (1 mentions)
Gemini 500, by Zeiss (1 mentions)
Tmb solution, by Merck Group (1 mentions)
Streptavidin hrp, by Merck Group (1 mentions)
Infinite 200 pro, by Tecan (1 mentions)
Jem arm200f, by JEOL (1 mentions)
Ex taq buffer, by Takara Bio (1 mentions)
Dpnii, by New England Biolabs (1 mentions)
Hinfi, by New England Biolabs (1 mentions)
Aminoallyl dutp cy5, by Jena Biosciences (1 mentions)
Topo xl pcr cloning kit, by Thermo Fisher Scientific (1 mentions)
Unmethylated lambda dna, by Promega (1 mentions)
Digoxigenin 16 dutp, by Roche (1 mentions)
Gotaq g2 flexi taq dna polymerase, by Promega (1 mentions)
16 protocols using biotin 16 dutp
1
Nascent chromatin capture assay protocol
2
CRISPR-based Detection of HPV and HBV
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The ssDNA and target DNA were procured from Bioneer. The specific ssDNAs used were [Thiol]-5′-ATATATATATA-3′-[Biotin] and [Thiol]-5′-ATATATATATA-3′. For the targets, the following DNAs were utilized: HPV16L1: 5′-GTGCTGCCATATCTACTTCA-3′, HPV18L1: 5′-TGTGTAGAAGCACATATTGT-3′, HBV: 5′-TTGGCTTTCAGTTATATGGATGATGTGGTA-3′, and BRCA-1: 5′-GAACAAAAGGAAGAAAATCA-3′. The crRNA and Cas12a were obtained from IDT, with the crRNAs used being HPV16L1 crRNA: 5′-UAAUUUCUACUAAGUGUAGAUUGAAGUAGAUAUGGCAGCAC-3′ and HPV18L1 crRNA: 5′-UAAUUUCUACUCUUGUAGAUACAAUAUGUGCUUCUACACA-3′. Terminal transferase, streptavidin-HRP, dATP, and TMB solution were sourced from Sigma-Aldrich. Biotin-16-dUTP was obtained from Jena Bioscience. The sample was characterized using field emission scanning electron microscopy (FE-SEM; Gemini500, Zeiss, Jena, Germany) and Cs_corrected field emission transmission electron microscopy (FE-TEM; JEM-ARM200F, Jeol, Tokyo, Japan); the microscopes were installed in the Center for University-wide Research Facilities (CURF) at Jeonbuk National University. Fluorescence spectra and absorbance spectra were collected using a multi-mode microplate reader (Tecan, TECAN infinite 200 PRO, Männedorf, Switzerland).
3
Chromosome Sorting and Painting Probe Generation
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Chromosome suspensions were sorted using an adaptation of a previously reported protocol60 (link) and a dual-laser cell sorter (MoFlo, Beckman Coulter), as performed at the Cambridge Resource Centre for Comparative Genomics (Cambridge, UK). Chromosome-specific painting probes were made by degenerate oligonucleotide primer PCR (DOP-PCR) amplification of flow-sorted chromosomes67 (link),68 (link). DOP-PCR amplified chromosome-specific DNAs were labeled during the secondary PCR by incorporating biotin-16-dUTP (Jena Bioscience) or digoxigenin-11-dUTP (Jena Bioscience). The PRO and PGO painting probes were generated as previously described69 (link).
4
Fluorescence In Situ Hybridization Protocols
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PRW LINE-like mapping was done in Jaén, Spain. 1 µg of PRW LINE-like DNA was labelled by nick translation using the biotin-NT-mix (Roche, Basel, Switzerland). The probe was then precipitated along with 50 μg yeast RNA and 50 μg salmon sperm DNA and redissolved in 50% formamide in 2 × SSC. Fluorescence in situ hybridization (FISH) was performed according to the published protocol39 (link) with listed modifications40 (link).
PRW Bel-Pao mapping was done in České Budějovice, Czech Republic. Both fragments (I and II, which do not contain the deleted region—see below) were labelled by PCR containing 0.04 mM each of dATP, dCTP and dGTP; 0.014 mM dTTP, 0.025 mM biotin-16-dUTP (Jena Bioscience, Jena, Germany), 1× ExTaq buffer (TaKaRa), 1–10 ng plasmid DNA, 1 μM of each primer and 2U DNA ExTaq Polymerase (TaKaRa) under the same conditions as described above. FISH was performed according to the published protocol41 (link), with signal amplification using Cy3-conjugated streptavidin (Jackson ImmunoRes. Labs. Inc, West Grove, PA, USA) at a dilution of 1:1000 with washing blocking buffer.
PRW Bel-Pao mapping was done in České Budějovice, Czech Republic. Both fragments (I and II, which do not contain the deleted region—see below) were labelled by PCR containing 0.04 mM each of dATP, dCTP and dGTP; 0.014 mM dTTP, 0.025 mM biotin-16-dUTP (Jena Bioscience, Jena, Germany), 1× ExTaq buffer (TaKaRa), 1–10 ng plasmid DNA, 1 μM of each primer and 2U DNA ExTaq Polymerase (TaKaRa) under the same conditions as described above. FISH was performed according to the published protocol41 (link), with signal amplification using Cy3-conjugated streptavidin (Jackson ImmunoRes. Labs. Inc, West Grove, PA, USA) at a dilution of 1:1000 with washing blocking buffer.
5
High-Throughput Chromatin Interaction Analysis
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Hi-C libraries of two biological replicates were generated utilizing the recently published in situ Hi-C methodology with minor modifications (45 (link)). The genomes were digested with either DpnII (NEB) or HinfI (NEB) and 5΄ overhangs were filled in with Biotin-16-dUTP (Jena Bioscience). HiC libraries were sequenced at the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) sequencing facility and the HudsonAlpha Genomic Services Laboratory. Paired reads were mapped to the genome, processed and matrix resolution was calculated as described previously (45 (link)). To call significant interactions, contact matrixes were first normalized by ICE and processed by Fit-Hi-C at 1 kb resolution for all interaction distances between 3 kb and 1 Mb (46 (link),47 (link)). Interaction calls from Fit-Hi-C were further filtered by their probability of occurring in a random generated list of interactions. Using this secondary filtering step, we obtained lists of interactions below a secondary false discovery rate (FDR < 0.001). To ensure Fit-Hi-C calls were accurate across replicates and/or experimental conditions, KR normalized reads (and further normalized by total sequencing depth) from HinfI and DpnII were plotted and the spearman correlation was calculated.
6
Single-Molecule DNA Construct Preparation
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Full sequences of all DNA molecules are given in Supplementary file 2 . All DNA molecules except ‘template 2’ in Figure 1 were prepared by ligating four or five DNA fragments, respectively: 1) ‘Cy5-biotin handle’, 2) ‘8.4 kb fragment’, [3) ‘Sequence of Interest’,] 4) ‘11.2 kb fragment’, and 5) ‘biotin handle’ (Figure 1—figure supplement 1b ). The ‘Cy5-biotin handle’ and ‘biotin handle’ were prepared by PCR methods in the presence of Cy5-modified and/or biotinylated dUTP (aminoallyl-dUTP-Cy5 and biotin-16-dUTP, Jena Bioscience). The ‘8kb-fragment’ and ‘11 kb fragment’ were prepared by PCR on Unmethylated Lambda DNA (Promega). These fragments were cloned into pCR-XL using the TOPO XL PCR cloning kit (Invitrogen) generating pCR-XL-11.2 and pCR-XL-8.4 (Ganji et al., 2016b (link)). The fragments were PCR amplified and then digested with BsaI restriction enzyme, respectively (Supplementary file 3 ). The ‘Sequence of Interest’ was made by PCR on different templates. Template two in Figure 1C -black and 1e was made from a digested fragment of an engineered plasmid pSuperCos-λ1,2 with XhoI and NotI-HF (van Loenhout et al., 2012 (link)). The digested fragment was further ligated with biotinylated PCR fragments on XhoI side and a biotinylated-Cy5 PCR fragment on the NotI-HF (Supplementary file 4 ). All the DNA samples were gel-purified before use.
7
Labeling Probes for Chromosomal FISH
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Probes for fluorescence in situ hybridization corresponding to CgiSat02, 03, 04, 05, 09, 17, 28, 37, 46 and 5S rDNA were labeled by PCR. The reactions contained 50 ng of DNA, 100 µM dATP, dGTP and dCTP, 65 µM dTTP, 2.5 mM MgCl2, 2.5 U GoTaq G2 Flexi Taq DNA polymerase, 1× GoTaq Flexi Reaction Buffer (all Promega, Madison, WI, USA), primers (1 µM each) and 35 µM biotin-16-dUTP (Jena Bioscience, Jena, Germany) for satDNAs or 35 µM digoxigenin-16-dUTP (Roche, Basel, Switzerland) for 5S rDNA, in 50 µL volumes. Nucleotide sequences of each primer pair used and PCR amplification conditions employed are presented in Table S5 . Probe purification was performed using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany), following the protocol within. Probes were checked on 1% agarose gel and the concentration of the purified probes was measured using a Qubit Fluorometer. 30 ng of probe was used per FISH experiment.
8
Fluorescence In Situ Hybridization of Meiocytes
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Fluorescence in situ hybridization was carried out on prophase 1 meiocytes prepared from immature flower buds that were fixed in ethanol/acetic acid (3:1), following the protocol by Ross et al.38 (link). The following BAC clones were used: F9H3, T19B17, T26N6, F4H6, T4B21, T1J1 (IGF and TAMU library)39 (link),40 (link). The BAC DNA clones were labeled with either digoxigenin-11-dUTP or biotin-16-dUTP (Jena Bioscience GmbH) by nick translation following the manufacturer’s protocol (Sigma-Aldrich). In situ hybridization was carried out according to Lysak et al., 2006 with separate denaturation of probes and chromosomes41 . Microscopy slides were examined with a Zeiss AxioScope A1 fluorescence microscope using small band pass filters for DAPI, FITC, and Cy3. Images were captured with a Nikon color DS-Ri2 camera using Nikon NIS-elements 4.60 software. Microscope images were further processed with Adobe Photoshop software.
9
Bonobo Y Chromosome Fluorescence Probe
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The WBY painting probe was prepared from flow-sorted bonobo Y chromosomes and labeled with biotin-16-dUTP (Jena BioScience) using DOP-PCR according to (Yang et al. 2009 ). Oligonucleotide probe (Pan32) (/5AmMC12/ATCTGTATAAACATGGAAATATCTACACCGCY) was prepared and labeled using Alexa Fluor oligonucleotide amine labeling kit (Invitrogen).
10
Enzyme-Mediated DNA Labeling Protocols
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Engen LbCas12a (#M0653T) and TdT (#M0315L) were obtained from New England Biolabs (Ipswich, MA, USA) as were the matching CoCl2 (2.5 mM stock) plus TdT buffer (final concentration: 50 mM potassium acetate, 20 mM Tris–acetate, and 10 mM magnesium acetate; pH 7.9). dTTP (ThermoFisher #R0171, Bleiswijk, The Netherlands). Fluorescein-12-dUTP (Jena Bioscience, Germany) (#NU-803-FAMX-L). Biotin-16-dUTP (Jena Bioscience, Germany) (#NU-803-BIO16-S). HybriDetect, Universal LFA Kit (Milenia Biotec, Gießen Germany). G-25 Sephadex Columns (Roche, #11273949001). Dideoxycytidine (GE Healthcare, USA) (#27-2061-01).
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