Electrophoretic mobility shift assay (EMSA) was carried out on a 1% native agarose gel, or a 4–16% acrylamide gel (Novex™ TBE Gels). PaP3 TerS from peak 2 was used in both assays. WT-TerS or the various mutants were mixed with Cy3-cos or Cy3-scr oligonucleotides and incubated at 37°C for 30 min. The protein:DNA mixture was resolved either on a 1% agarose gel (49 (link)) or a 4–16% acrylamide gel (Novex™ TBE Gels) in the presence of 0.5× TBE buffer (45 mM Tris base, 45 mM Boric Acid, 1 mM EDTA) at 4°C. The Cy3 signals were measured using a ChemiDoc MP (Bio-Rad) at 602 nm. The intensity of the bands was quantified using ImageJ (49 (link)). The fraction of TerS bound to DNA was calculated by dividing the total intensity of all bands in each lane by that of the bands representing protein–DNA complexes. The plot was generated using GraphPad Prism 8 based on three independent experiments.
Tbe gel
Manufactured by Thermo Fisher Scientific
Sourced in United States
TBE gel is a type of agarose gel used for the separation and analysis of DNA and RNA molecules in molecular biology and biochemistry. It functions as a medium for the electrophoretic separation of nucleic acid samples.
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Lab products found in correlation
Sybr gold, by Thermo Fisher Scientific (5 mentions)
Hiseq 2000, by Illumina (4 mentions)
P 30 column, by Bio-Rad (4 mentions)
Nextseq 500, by Illumina (4 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (4 mentions)
Turbo dnase, by Thermo Fisher Scientific (3 mentions)
Lightshift chemiluminescent emsa kit, by Thermo Fisher Scientific (3 mentions)
2100 bioanalyzer, by Agilent Technologies (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Chemidoc mp, by Bio-Rad (2 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (2 mentions)
Hiseq 4000, by Illumina (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Biodyne b nylon membrane, by Thermo Fisher Scientific (2 mentions)
X tremegene 9, by Roche (2 mentions)
Pcmv6 entry, by OriGene (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Sybr gold stain, by Thermo Fisher Scientific (2 mentions)
Phix control v3 dna, by Illumina (2 mentions)
54 protocols using tbe gel
1
DNA-Binding Dynamics of TerS Mutants
2
INO80-Mediated Nucleosome Sliding Dynamics
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Nucleosome preparation and sliding by INO80 was performed under conditions as reported earlier19 (link). Sliding for in the presence of various [INO80] as reported in Fig. 2f (for 1 minute) and Extended Data Fig. 7 (for 1 minute and 2.5 minutes) was performed in 10 μl reaction volumes containing 8 nM nucleosomes (nucleosomes formed on both constructs in the pair were present in equimolar proportion), 2 mM ATP, 24 mM tris-HCl pH 7.5, 43 mM KCl, 2.86 mM MgCl2, 0.55% glycerol and indicated concentration of INO80. The mixture was incubated without ATP at 30 °C for 7 minutes. After addition of ATP, the reaction was allowed to proceed for 1 minute at 30 °C, and was then quenched by the addition of lambda DNA and ADP to final concentrations of 66.7 μg/ml and 20 mM respectively. For all sliding experiments reported in Extended Data Fig. 6b (timecourse of INO80 sliding), conditions were the same except incubations prior to ATP addition and the subsequent sliding reaction were carried out at room temperature. The reaction was continued for the indicated amounts of time in presence of saturating INO80 prior to quenching. Quenched reactions were loaded on to 6% TBE gels (Invitrogen) in presence of 10% glycerol and run at 150 V for 1.5 hrs. The gel was imaged separately for Cy3 and Cy5 fluorescence.
3
Protein-DNA Binding Assay Protocol
4
INO80-Mediated Nucleosome Sliding Dynamics
5
Helraiser Transposase Binding to DNA
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Binding of the Helraiser transposase to various DNA oligonucleotides was measured by EMSA using 6% TBE gels (Invitrogen). Purified protein at 15–250 nM was incubated for 30 min at room temperature in binding buffer (50 mM Tris pH 7.5, 100 mM NaCl, 10 mM MgCl2, 0.5 mM EDTA, 1 mM TCEP) with 50 nM 6-FAM-labelled oligonucleotides. To test whether the addition of a nonhydrolyzable ATP analogue could lock Helraiser helicase domain into an active conformation and facilitate DNA binding, 1 mM AMP–PNP was added to some of the binding reactions. After addition of DNA gel loading solution (Quality Biological, INC), samples were run on 6% TBE gels and visualized.
6
Small RNA-Seq Analysis of Plant Seedlings
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Total RNA extraction was performed by using TRIzol reagent (Invitrogen, #15596026) from 7-day-old seedlings, and dissolved in DEPC-treated H2O. Total RNAs were used for library preparation with Real Seq Bioscience RealSeqR-AC Kit (500–00012). The final library products were further purified using 6% polyacrylamide gel (Novex™ TBE Gels, EC6265BOX). The 145–160nt products were excised from the gel for sequencing (single end 50 bp) on a NextSeq 2000 machine (Illumina). The small RNA sequencing data were trimmed using Trimmomatic (v.0.39) and then mapped to the pseudo-genome sequence with insertion of 2 × 180 or 5 × 180 in TAIR10 and called small RNA using ShortStack version 3.8.5 (44 (link)) with parameter setting "–mincov 1rpm –pad 75 –mismatches 0 –nohp.
7
Tethered Conformation Capture of Hypoxic Cells
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For two replicates, 90 million cells were grown in normoxic (20% O2) or in hypoxic (1% O2) conditions (Ruskinn InVivo2 400 hypoxia incubator) for 8 h. Tethered conformation capture (TCC) was performed as described (3 (link)). Digestions were performed using MboI (New England Biolabs), DNA was sheared with Bioruptor Next-Gen (Diagenode) for 30–35 cycles, library preparations continued as described (3 (link)) and products were amplified for 15 cycles, size-selected (225–550 bp) from 10% TBE gels (Life Technologies) and sequenced using Illumina HiSeq 2000 at EMBL Genomics Core Facility (Heidelberg, Germany).
8
GRO-seq Library Preparation for Hypoxia
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GRO-seq libraries were prepared from four biological replicates of control or hypoxia-treated HUVECs according to the protocol described (3 (link)). Briefly, 5–10 millions of nuclei were collected from each sample, run-on reactions were performed in the presence of Br-UTP and RNA was extracted with Trizol LS Reagent (Life Technologies, Carlsbad, CA, USA). RNA was DNAse treated and fragmented using TURBO DNase and RNA fragmentation reagents (Life Technologies), purified using P-30 columns (Bio-Rad, Hercules, CA, USA) and dephosphorylated using PNK (Enzymatics, Y904L). Labeled RNA fragments were captured using anti-BrdU beads (sc-32323 AC, SantaCruz Biotech, Santa Cruz, CA, USA). Poly(A)-tailing and reverse transcription were performed as described (3 (link)). Libraries were amplified using 12–16 cycles, size-selected (180–350 bp) from 10% TBE gels (Life Technologies) and sequenced using Illumina HiSeq 2000.
9
Generating Strand-Specific RNA-Seq and ChIP-Seq Libraries
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Sequencing libraries were prepared from recovered DNA (ChIP) or generated cDNA (RNA) by blunting, A-tailing, and adapter ligation as previously described using barcoded adapters (NextFlex; Bioo Scientific) (Gosselin et al, 2014 (link), 2017 (link)). Before final PCR amplification, RNA-seq libraries were digested by 30 min of incubation at 37°C with Uracil DNA Glycosylase (final concentration of 0.134 units per μl of library volume; UDG, Enzymatics G5010L) to generate strand-specific libraries. Libraries were PCR-amplified for 12–15 cycles and size selected for fragments (200–400 bp for ChIP-seq, 200–500 for RNA-seq) by gel extraction (10% TBE gels, Life Technologies EC62752BOX). RNA-seq and ChIP-seq libraries were single-end sequenced on an Illumina HiSeq 4000 (Illumina) according to manufacturer’s instruction.
10
DNA-Binding Affinity Assay for Taq Polymerase
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Dissociation constant Kd (DNA) was measured using a gel mobility-shift assay that employs a 5′ 33P-labeled hairpin template (5′-CTCCAGACACGACGCAGTTGCCCGATGGTCGACGTTCGCGAAAGCGAACGTCGACCATCGGGCAACT-3′) blocked at the 3′ end with dideoxy TMP. The radiolabeled hairpin (0.25 nM) was incubated with varying concentrations of wild-type or mutant Taq DNA polymerases (0.5–1000 nM; bracketing the predicted Kd) in 1× Taq buffer at 37°C for 30 min. DNA-protein complexes were run on 10% non-denaturing TBE gels (Life Technologies) in 0.5× TBE buffer. Gels were dried down and exposed to film. The fraction of DNA bound was quantified by densitometry using AlphaView SA software (Alpha Innotech), and then plotted against enzyme concentration to determine Kd values by interpolation.
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