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| Status |
Public on Oct 08, 2019 |
| Title |
Ecoli_Chr1 |
| Sample type |
SRA |
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| Source name |
E.coli cells
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| Organism |
Escherichia coli |
| Characteristics |
strain: expressing Hta
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| Treatment protocol |
T. acidophilum cells self-lyse at pH>6 (Robb et al. 1995). Pellets were therefore directly re-suspended in ice-cold MNase digestion buffer (10mM Tris, 5mM Ca2+, pH8) and homogenized by 20 passages through a Dounce homogenizer, on ice. Unfixed crude lysates were digested in the presence of 4U/mL of MNase (Thermo Fisher) at 37°C. Digestion was stopped by addition of EDTA to a final concentration of 20mM. Samples were incubated for an additional 30min at 37°C in the presence of RNAse A to a final concentration of 0.5mg/mL and then overnight at 65°C in the presence of SDS (1%) and proteinase K (125µg/mL). Undigested lysate was incubated as above but without addition of enzyme. Undigested DNA was then sonicated using a Covaris S220 sonicator with the following settings: peak power: 175, duty: 10, cycle/burst: 200 for 430s (target size: 150bp).
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| Growth protocol |
T. acidophilum strain 122-1B2 was obtained from DSMZ (https://www.dsmz.de/) and cultured using the medium described in (Searcy and Stein 1980), supplemented to a final concentration of 2g/L yeast extract (BD Biosciences). The medium was boiled for five minutes and allowed to cool to 58°C before inoculation. Cultures were incubated at 59°C with shaking (90rpm) in an INFORS Thermotron incubator.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted by phenol chloroform extraction and precipitated with ethanol.
For MNase digestion experiments paired-end reads were prepared using the NEBNext Ultra II DNA Library Prep Kit.
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| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Mnase digested chromatin DNA
Zscore_Merged_replicates_Ecoli_ChrDig_40_65bp.bw
Zscore_Merged_replicates_Ecoli_ChrDig_70_100bp.bw
Zscore_Merged_replicates_Ecoli_ChrDig_total.bw
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| Data processing |
Basecalls performed using CASAVA 1.8.4
Reads were aligned to NC_002578.1 T.acidophilum genome assembly or to E.Coli C600 genome (CP031214) obtained from ncbi, using Bowtie2
Paired-end 100bp reads were first trimmed using BBDuk v37.64 (parameters: ktrim=r, k=21, hdist=1, edist=0, mink=11, qtrim=rl, trimq=30, minlength=10, ordered=t, qin=33) and then merged using BBmerge v37.64 (parameters: mininsert=10, mininsert0=10).
The merged reads were mapped to the T. acidophilum DSM1728 genome (GCA_000195915.1) or to E.Coli C600 genome (CP031214) using Bowtie2 (-U).
Coverage tracks were computed using deeptools bamCoverage (RPGC normalization, effective genome size: 1564906bp), measuring coverage for reads of sizes 40-65bp and 70-100bp separately. Reads from the sonicated DNA control samples were mapped in paired-end mode. Coverage was computed independently of read size and smoothed over 10kbp to avoid introducing noise from the input into the MNase signal. Genome-wide coverage of MNase-digested samples was then divided by its corresponding undigested DNA sample to remove bias in coverage associated with replication. Lastly, coverage was converted into a Z-score using the scale function in R.
Genome_build: T.acidophilum : NC_002578.1 ; E.coli : CP031214
Supplementary_files_format_and_content: Bigwig
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| Submission date |
Oct 08, 2019 |
| Last update date |
Oct 09, 2019 |
| Contact name |
Tobias Warnecke |
| E-mail(s) |
molecular.systems.laboratory@gmail.com
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| Organization name |
Imperial College
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| Street address |
Du Cane Road
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| City |
London |
| ZIP/Postal code |
W12 0NN |
| Country |
United Kingdom |
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| Platform ID |
GPL18133 |
| Series (2) |
| GSE127728 |
The DNA-binding protein HTa from Thermoplasma acidophilum is an archaeal histone analog |
| GSE138576 |
The DNA-binding protein HTa from Thermoplasma acidophilum is an archaeal histone analog (Mnase-seq and EMSA-seq) |
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| Relations |
| BioSample |
SAMN12993502 |
| SRA |
SRX6964052 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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