The “601” nucleosome-positioning DNA sequence was used for nucleosomal template preparation (Lowary & Widom, 1998 (link)). The DNA template was amplified by PCR and fluorescently labeled using a Cy-5 conjugated reverse primer. The PCR product was purified with a PCR purification kit (QIAGEN) before nucleosome reconstitution. For nucleosome assembly, the DNA template was mixed with recombinant histone octamer in a 1:3 M ratio and a twofold DNA mass of sheared salmon sperm DNA (Ambion) and the mixture was then dialyzed in dialysis buffers (10 mM Tris–HCl, pH 7.5, 0.1% NP-40, 0.2 mM EDTA, and 5 mM 2-mercaptoethanol) containing various salt concentrations (2, 1.5, 1, 0.75, 0.5 M, and 10 mM NaCl, respectively) from high to low salt stepwise as described previously (Hsieh et al, 2015 (link)).
Sheared salmon sperm dna
Manufactured by Thermo Fisher Scientific
Sourced in United States
Sheared salmon sperm DNA is a high-quality DNA preparation derived from salmon sperm. It is designed to serve as a standard or control in various molecular biology applications, such as DNA quantification, amplification, and hybridization experiments.
Automatically generated - may contain errors
Lab products found in correlation
Nupage novex bis tris precast gel, by Thermo Fisher Scientific (4 mentions)
Igepal ca 630, by Merck Group (3 mentions)
Desthiobiotin datp, by Jena Biosciences (3 mentions)
T4 ligase, by Thermo Fisher Scientific (3 mentions)
Dynabeads myone c1, by Thermo Fisher Scientific (3 mentions)
Laemmli buffer, by Merck Group (2 mentions)
Microspin g 50 column, by GE Healthcare (2 mentions)
T4 kinase, by Thermo Fisher Scientific (2 mentions)
Dna polymerase, by Thermo Fisher Scientific (2 mentions)
Klenow fragment, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Dynabeads myone streptavidin c1, by Thermo Fisher Scientific (2 mentions)
Human placental dna, by Merck Group (2 mentions)
Ssofast evagreen supermix, by Bio-Rad (2 mentions)
Cfx96, by Bio-Rad (2 mentions)
Pcr purification kit, by Qiagen (1 mentions)
G50 spin column, by GE Healthcare (1 mentions)
G 50 columns, by GE Healthcare (1 mentions)
T4 polynucleotide kinase, by Thermo Fisher Scientific (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
26 protocols using sheared salmon sperm dna
1
Nucleosome Reconstitution from 601 Sequence
2
ChIP Re-ChIP Protocol for Chromatin Analysis
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Chromatin for ChIP Re-ChIP experiments was prepared as described in Negre et al. (59 (link)). Six separate ChIP reactions were set up for the first IP of a single ChIP Re-ChIP experiment. Shortly, protein A or A/G Sepharose (GE Healthcare) was pre-blocked with 1 mg/ml BSA and 1 mg/ml sheared salmon sperm DNA (Ambion). Antibodies were pre-bound to the beads at 4°C for at least 6 h in 900 μl Dilution IP buffer (16.7 mM Tris pH 8, 1.2 mM EDTA pH8, 167 mM NaCl, 0.01% SDS, 1.1% Triton X100), chromatin was added and rotated at 4°C overnight. Beads were washed with 1 ml of Dilution IP buffer (2x), TSE buffer (1x) (20 mM Tris pH8, 2 mM EDTA pH8, 500 mM NaCl, 1% Triton X100, 0.1% SDS), LiCl buffer (1x) (100 mM Tris pH8, 500 mM LiCl, 1% deoxycholic acid, 1% NP40) and TE buffer (2x). Six ChIP reactions were combined and eluted for 20 min at 65°C in elution buffer (50 mM NaHCO3, 140 mM NaCl, 1% SDS). Eluted material was added to antibody pre-bound to beads in Dilution IP buffer (see above) and incubated at 4°C overnight. Beads were washed as described above, DNA was eluted and purified as described (59 (link)) and used in qRT-PCR experiments.
3
Affinity Purification of Telomeric Proteins
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25 μg of forward and reverse sequence oligonucleotides (Table 1 ) were diluted in annealing buffer (20 mM Tris–HCl, pH 7.5, 10 mM MgCl2, 100 mM KCl), denatured at 95°C, and annealed by cooling. Annealed double‐strand oligonucleotides were incubated with 100 units T4 kinase (Life Technologies) for 2 h at 37°C followed by incubation with 20 units T4 ligase overnight. Concatenated DNA strands were purified using phenol–chloroform extraction. Following biotinylation with desthiobiotin‐dATP (Jena Bioscience) and 60 units DNA polymerase (Thermo Scientific), the biotinylated probes were purified using microspin G‐50 columns (GE Healthcare). Telomeric or control DNA was immobilized on 375 μg paramagnetic streptavidin beads (Dynabeads MyOne C1, Thermo Scientific) on a rotation wheel for 30 min at room temperature. Subsequently, baits were incubated with 40 μg nuclear extract in PBB buffer (150 mM NaCl, 50 mM Tris–HCl pH 7.5, 5 mM MgCl2, 0.5% Igepal CA‐630 (Sigma) and 1 mM DTT) while rotating for 2 h at 4°C. 20 μg sheared salmon sperm DNA (Ambion) was added as a competitor for DNA binding. After three washes with PBB buffer, bound proteins were eluted in 2× Laemmli buffer (Sigma‐Aldrich), boiled for 5 min at 95°C and separated on a 4–12% NuPAGE Novex Bis–Tris precast gel (Thermo Scientific).
4
TERT Promoter DNA-Protein Interaction
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Biotinylated DNA baits consisting of the 2nd Sp1 binding site in the TERT promoter or a mutated sequence were prepared as previously described [51 (link)]. For primer sequences see Table 4
5
Lithium Acetate Transformation of PCR Products
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CEN5 was deleted as previously described [32 (link)]. Lithium acetate transformation of PCR products with at least 70 bp of homology to the targeted gene was used for strain construction. Briefly, strains to be transformed were inoculated in liquid YPAD (10g/L yeast extract, 20g/L bactopeptone, 0.04g/L adenine, 0.08g/L uridine, 20g/L dextrose) and grown at 30°C for 16–18 h. Cultures were then diluted 1:166 in YPAD and grown at 30°C for 3–4 h. Cells were washed with water, then TELiAc (10mM Tris pH 7.5, 1mM EDTA, 100mM LiAc) and incubated in TELiAc with transformation DNA and 50μg sheared salmon sperm DNA (Ambion) for 30 min. 4 volumes PLATE mix (40% PEG, 10mM Tris pH 7.5, 1mM EDTA, 100mM LiAc) was then added and the transformation mix was incubated for 16–18 h at 20–24°C. Transformations were heat shocked at 42°C for 1 h, then plated on selective media with the exception of NAT1 marker transformations, which were recovered on non-selective media for 6 h prior to replica plating to selective media containing 400 μg/ml nourseothricin (Werner BioAgents). Strains were checked by PCR of genomic DNA.
6
Telomeric DNA Pulldown Assay
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Forward and reverse sequence oligonucleotides (25 μg) (see Supplementary Table 3 ) were diluted in annealing buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 100 mM KCl), denatured at 95 °C and annealed by cooling. Annealed double-stranded oligonucleotides were incubated with 100 units T4 kinase (Life Technologies) for 2 h at 37 °C followed by incubation with 20 units T4 ligase overnight. Concatenated DNA strands were purified using phenol–chloroform extraction. Following biotinylation with desthiobiotin-dATP (Jena Bioscience) and 60 units DNA polymerase (Thermo), the biotinylated probes were purified using microspin G-50 columns (GE Healthcare). Telomeric or control DNA was immobilized on 500 μg paramagnetic streptavidin beads (Dynabeads MyOne C1, Life Technologies) on a rotation wheel for 30 min at room temperature. Subsequently, baits were incubated with 400 or 800 μg (frog) of nuclear extract in PBB buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 5 mM MgCl2, 0.5% Igepal CA-630 (Sigma)) while rotating for 1.5 h at 4 °C. Sheared salmon sperm DNA (10 μg; Ambion) was added as a competitor for DNA binding. After three washes with PBB buffer, bound proteins were eluted in 1 × LDS sample buffer supplemented with 0.1 M DTT, boiled for 10 min at 70 °C and separated on a 10% NuPAGE Novex Bis-Tris precast gel (Life Technologies).
7
Telomere-Binding Interactome Identification
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Telomere pull-downs were done as previously described (43 (link)). In brief, chemically synthesized oligonucleotides with either TTAGGG or TGTGAG repeats were annealed with their complementary oligonucleotides. The dsDNA was phosphorylated with 100 units PNK (Thermo) for 2 h at 37°C and ligated overnight with 20 units T4 ligase (Thermo) at room temperature (RT). The mixture was cleaned by chloroform phenol extraction and the purified DNA was incubated with biotin-dATP (Jena Biosciences) and 60 units Klenow fragment (Thermo) at 37°C overnight. The DNA was re-buffered using a G50 Spin column (GE Healthcare) according to the manufacturer’s instructions. Around 25 μg biotinylated DNA was immobilized on Streptavidin Dynabeads MyOne C1 (Thermo) and incubated with trypanosome PCF whole cell lysate obtained by lysis in modified RIPA buffer [50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% Igepal CA-630, 0.1% Sodium Deoxycholate, Protease inhibitor cocktail (Roche)]. The binding reaction was performed in protein binding buffer (PBB) (150 mM NaCl, 50 mM Tris–HCl pH 7.5, 5 mM MgCl2, 0.5% Igepal CA-630) in the presence of 10 μg sheared salmon sperm DNA (Ambion) at 4°C for 2 h under slight agitation. Unbound proteins were washed with PBB three times and the bound fraction eluted with 1× lithium dodecyl sulfate (LDS) buffer (Thermo).
8
Telomere-Binding Protein Pulldown Assay
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Twenty five microgram of forward and reverse oligonucleotides of telomeric, variant repeat or control sequences (Supplementary Table S1 ) were denatured at 80°C and annealed by cooling. Double-stranded oligonucleotides were then polymerized using 50 U T4 polynucleotidekinase (Thermo Scientific) and 80 U T4 DNA ligase (NEB), biotinylated with desthiobiotin-dATP (Jena Bioscience) by Klenow fragment (Thermo Scientific) and purified using G-50 columns (GE Healthcare). Chemically synthesized DNA was immobilized on 0.5 mg streptavidin-coated magnetic beads (Dynabeads MyOne Streptavidin C1, Invitrogen) for 15 min at RT and incubated with 400–800 μg protein lysates diluted in PBB buffer (150 mM NaCl, 50 mM Tris–HCl pH 7.5, 10 mM MgCl2, 0.5% IGEPAL CA-630, 1 μM Pepstatin, 1 μg/ml Leupeptin and 0.5 mM PMSF) for 1.5 h on a rotation wheel at 4°C. Sheared salmon sperm DNA (16.7 μg; Ambion) was added as a competitor. After three washes with PBB buffer, bound proteins were eluted in LDS sample buffer supplemented with 0.1 M DTT, boiled for 10 min at 70°C and separated on a 4–12% NuPAGE Novex Bis–Tris precast gel (Life Technologies) for 50 min at 180 V in 1× MES buffer.
9
DNA Bait Pulldown Proteomics
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Biotinylated DNA baits were immobilized on magnetic streptavidin beads (MyOne C1 Streptavidin Dynabeads, Thermo) for 30 min at room temperature on a rotation wheel. DNA baits were incubated with 500 μg of protein extracts diluted in PBB buffer (150 mM NaCl, 50 mM Tris–HCl pH 7.5, 0.5% IGEPAL CA‐630, 5 mM MgCl2, 1 mM DTT) using 20 μg sheared salmon sperm DNA as a competitor (Thermo). Protein extracts were incubated with DNA baits for 1 h at 4°C on a rotation wheel. A fraction of the samples was treated with five units of RNase H (NEB) and 20 μg of RNase A (Thermo Scientific) during incubation. DNA baits were washed three times with PBB buffer, and bound proteins were eluted by heating for 10 min at 75°C in 1× NuPAGE LDS buffer (Thermo) supplemented with 100 mM DTT.
10
Fluorescent FISH Probe Generation
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Probes for FISH were generated using a modified conventional PCR: the reaction mix with a final volume of 25 μl contained 0.1 mM each of unlabelled dATP, dCTP and dGTP and 0.03 mM of dTTP; 0.5 μl one fluorophore conjugated dUTP (cyanine 3-dUTP (Enzo), cyanine 5-dUTP (Enzo) or fluorescein-12-dUTP (Thermo Fisher Scientific)); 1× Taq-buffer; and 0.625 U GoTaq DNA Polymerase (Promega). Each PCR amplification was performed using 0.5 μg of genomic DNA template from testes, 34 PCR cycles and a 30-s extension step to obtain appropriately sized probes for FISH. After cycling the reaction, 25 μl PCR mix was combined with 5 μl sheared salmon sperm DNA (1 mg ml−1; Thermo Fisher Scientific), 3 μl 3 M sodium acetate, pH 5.2 and 80 μl 100% cold ethanol, and kept overnight at −20 °C for probe precipitation. After spinning and supernatant removal, the pellet was dissolved in 25–30 μl of 50% formamide and stored at −20 °C before use.
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