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| Status |
Public on Feb 10, 2020 |
| Title |
Rep4_ec_none_INO80_80 |
| Sample type |
SRA |
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| Source name |
in vitro reconstituted chromatin
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| Organism |
Escherichia coli |
| Characteristics |
remodeler: INO80
barrier: none
histone-dna ratio: 0.8
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| Treatment protocol |
Genome-wide remodeling reaction: All remodeling reactions, except Chd1-containing reactions, were performed at 30 °C in 100 µL with final buffer conditions of 26.6 mM HEPES-NaOH pH 7.5, 1 mM Tris-HCl pH 7.6, 85.5 mM NaCl, 8 mM KCl, 10 mM ammonium sulfate, 10 mM creatine phosphate (Sigma), 3mM MgCl2, 2.5 mM ATP, 0.1 mM EDTA, 0.6 mM EGTA, 1 mM DTT, 14% glycerol, 20 ng/µL creatine kinase (Roche Applied Science). Chd1-containing reactions were performed in 26.6 mM HEPES-NaOH pH 7.5, 1 mM Tris-HCl pH 7.6, 50 mM NaCl, 10 mM creatine phosphate (Sigma), 3 mM MgCl2, 2.5 mM ATP, 0.1 mM EDTA, 0.6 mM EGTA, 1 mM DTT, 14% glycerol, 20 ng/µL creatine kinase. Usually, 10 nM of remodeling enzyme (50 nM Chd1/FACT), 40 nM Reb1 and 20 U of BamHI was added. Before full-length Chd1 (in high-salt buffer) was added to the reaction, it was diluted together with FACT to low salt buffer. For that, full-length Chd1 and purified FACT was mixed in a 1.2:1 ratio in high salt buffer (300 mM NaCl, 20 mM Na·HEPES pH 7.4, 10% (v/v) glycerol, 1 mM DTT), incubated for 5 minutes on ice and then diluted to 30 mM NaCl final concentration.
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| Growth protocol |
Salt Gradient Dialysis: For low, medium and high assembly degrees, 10 µg of plasmid library DNA (pGP546, S. pombe or E. coli) was mixed with ~2, 4 or 8 µg of Drosophila embryo histones, respectively, in 100 µl assembly buffer (10 mM Tris-HCl, pH 7.6, 2 M NaCl, 1 mM EDTA, 0.05% IGEPAL CA630, 0.2 µg BSA). Samples were transferred to Slide-A-lyzer mini dialysis devices, which were placed in a 3 L beaker containing 300 ml of high salt buffer (10 mM Tris-HCl pH 7.6, 2 M NaCl, 1 mM EDTA, 0.05% IGEPAL CA630, 14.3 mM β-mercaptoethanol), and dialyzed against a total of 3 L low salt buffer (10 mM Tris-HCl pH 7.6, 50 mM NaCl, 1 mM EDTA, 0.05% IGEPAL CA630, 1.4 mM β-mercaptoethanol) added continuously via a peristaltic pump over a time course of 16 h under stirring conditions. β-mercaptoethanol was added freshly to all buffers. After complete transfer of low salt buffer, samples were additionally dialyzed against 1 L low salt buffer for 1 h at room temperature. DNA concentration of the SGD chromatin preparations was estimated with a DS-11+ spektrophotometer (Denovix) and if necessary stored at 4 °C for several weeks. To estimate the extend of the assembly degree, an aliquot of the sample was subjected to MNase digestion (as described below) for MNase-ladder read out.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Remodeling was started by adding 10 µl SGD chromatin and terminated by adding 0.8 U apyrase followed by incubation at 30 °C for 30 minutes.
For library preparation, approximately 10 - 50 ng of mononucleosomal DNA was incubated with 1.25 U Taq polymerase (NEB), 3.75 U T4 DNA polymerase (NEB) and 12.5 U T4-PNK (NEB) in 1x ligation buffer (B0202S, NEB) for 15 minutes at 12 °C, 15 minutes at 37 °C and 20 minutes at 72 °C. To ligate NEBNext Adaptors (0.75 µM final concentration, from NEBNext Multiplex Oligos Kit) to the DNA, samples were incubated with T4 DNA ligase (NEB) at 25 °C for 15 minutes, followed by incubation with 2 U USER enzyme (NEB) for 10 minutes at 37°C. Fragments were purified using 2 volumes AMPure XP beads (Beckman Coulter) and amplified for 8-10 cycles using NEBNext Multiplex Oligos, Phusion High-Fidelity DNA Polymerase (1 U, NEB), deoxynucleotide solution mix (dNTP, 2.5 mM, NEB) and Phusion HF Buffer (1x, NEB). The following protocol was applied for amplification: 98 °C for 30 s, 98°C for 10s, 65 °C for 30s, 72 °C for 30s with a final amplification step at 72 °C for 5 minutes. The Qubit dsDNA HS Assay Kit (Invitrogen) was used to assess the DNA content of 1 µL PCR reaction. DNA was purified from agarose gels via PureLink Quick Gel Extraction Kit, measured again with Qubit dsDNA HS Assay Kit and diluted to final concentration of 10 nM (calculation based on the assumption that the DNA fragment length is 272 bp, i. e., 147 bp nucleosomal DNA and 122 bp sequencing adaptor). Diluted samples were pooled according to sequencing reads (~6 Mio reads/ sample). The final pool was quantified by BioAnalyzer (Agilent) and analyzed on an Illumina HiSeq 1500 in the 50 bp single-end mode (Laboratory for Functional Genome Analysis, LAFUGA, LMU Munich).
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| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina HiSeq 1500 |
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| Description |
in vitro
plasmid DNA
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| Data processing |
fastq files were aligned to the SacCer3 (R64), EF2 or E. coli strain B (REL606) genomes using Bowtie (Lengmead et al. 2009). Multiple matches were omited.
BAM files were imported into R Studio (RStudio Team, 2015) using GenomicAlignments (Lawrence et al. 2013). Then, reads were shifted by 73 bp to cover the nucleosome dyad and extended to 50 bp. Genome coverage was caluculated.
Coverage files were either aligned to in vivo +1 nucleosome positions, Reb1 SLIM-ChIP hits (Gutin et al. 2019) or Reb1 PWM hits (Badis et al. 2008), or BamHI cut sites. Signal was normalized per gene in a 2000 bp window around the alignment point.
Genome_build: SacCer3, EF2, Ecoli strain B - REL606
Supplementary_files_format_and_content: bw (big wig) files were generated from genome coverage files using rtracklayer (Lawrence et al. 2009).
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| Submission date |
Nov 18, 2019 |
| Last update date |
Feb 11, 2020 |
| Contact name |
Elisa Oberbeckmann |
| E-mail(s) |
elisa.oberbeckmann@med.uni-goettingen.de
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| Phone |
+49 551 39 65975
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| Organization name |
University Medical Center Göttingen
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| Department |
Molecular Biology
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| Lab |
Oberbeckmann
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| Street address |
Justus-von-Liebig Weg 11
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| City |
Göttingen |
| ZIP/Postal code |
37077 |
| Country |
Germany |
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| Platform ID |
GPL20045 |
| Series (1) |
| GSE140614 |
Ruler elements in chromatin remodelers set nucleosome array spacing and phasing |
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| Relations |
| BioSample |
SAMN13322813 |
| SRA |
SRX7176849 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4175579_Rep4_ec_none_INO80_80.bw |
10.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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