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| Status |
Public on Mar 11, 2019 |
| Title |
S_Mf_3 |
| Sample type |
SRA |
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| Source name |
Methanothermus fervidus cells
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| Organism |
Methanothermus fervidus |
| Characteristics |
growth phase: stationary phase
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| Treatment protocol |
~0.5 g of pellet were thawed and resuspended in 9 ml of 1x PBS. Cells were fixed by adding a fixation solution (100mM NaCl, 50mMTris-HCl pH 8.0, 10% formaldehyde) to a final concentration of 1% formaldehyde for 10 minutes (slow rotation) at room temperature. 140mM Glycine for 10 minutes at room temperature (rotating slowly) inhibited formaldehyde and avoided over-fixation. Formaldehyde and glycine residues were washed away by centrifugation (4000 rpm - 5 min - 4C) then bacteria were washed with 10 ml chilled 1x PBS, twice. The archaeal cell pellet was resuspended in 20 ml of chilled 1x PBS. The cell suspension thus obtained was passaged twice through a TS Series French press (Constant Systems) at 15 kpsi and then spun down at 4000 rpm for 15 minutes at 4C before proceeding with cell lysis.The pellet was resuspended in 500 ul of lysis buffer (10mM NaCl, 10mM Tris-HCl pH 7.4, 3mM MgCl2, 0.5% NP-40, 1x Pi, 0.15mM Spermine, 0.5mM Spermidine), transferred in a new microcentrifuge tube and incubated for 20 minutes on ice. The lysate was spinned down and the pelletwas washed with 500 ul of -CA buffer (15mM NaCl, 10mM Tris-HCl pH 7.4, 60mM KCl, 1x Pi, 0.15mM Spermine, 0.5mM Spermidine) without resuspending. The washed pellet was finally resuspended in 250 ul of +CA Buffer (15mM NaCl, 10mM Tris-HCl pH 7.4, 60mM KCl, 1mM CaCl2, 0.15mM Spermine, 0.5mM Spermidine) to a uniform suspension. 50 ul of this suspension were digested with the wanted concentration of micrococcal nuclease (MN) for 10 minutes (20 minutes for cells in stationary phase) at room temperature and finally blocked with a STOP solution containing calcium-chelating agents (100mM EDTA, 10mM EGTA). Each sample was further diluted with -CA buffer (without Pi, spermine and spermidine) and treated with 10% SDS and 150ng/ml proteinase K overnight at 65C shaking (~500 rpm).
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| Growth protocol |
Flash-frozen pellets of M. fervidus harvested in late exponential and stationary phase were purchased from the Archaeenzentrum in Regensburg (via Harald Huber).
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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 single-end reads were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina.
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| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina MiSeq |
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| Description |
Mnase digested DNA
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| Data processing |
Basecalls performed using CASAVA 1.8.4
Reads were first trimmed with trimgalore to remove adapters and low quality bases.
The trimmed reads were then mapped to the Methanothermus fervidus DSM 2088 genome (GCA_000166095.1) using Bowtie2 (--no-discordant --no-mix). Multimappers were filtered out to retain only reads mapping uniquely.
Coverage tracks were computed using the coverage function in the rtracklayer package for R.
Genome_build: NC_014658.1
Supplementary_files_format_and_content: bigWig
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| Submission date |
Mar 01, 2019 |
| Last update date |
Mar 11, 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 |
GPL26241 |
| Series (2) |
| GSE127678 |
The role of archaeal histones in gene expression - a synthetic biology perspective [Methanothermus fervidus] |
| GSE127680 |
The role of archaeal histones in gene expression - a synthetic biology perspective |
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| Relations |
| BioSample |
SAMN11040597 |
| SRA |
SRX5447129 |
| 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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