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| Status |
Public on Nov 27, 2019 |
| Title |
HrrA binding 9h after heme addition |
| Sample type |
SRA |
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| Source name |
precultures
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| Organism |
Corynebacterium glutamicum |
| Characteristics |
time point: 9h
medium: CGXII
heme concentration: 4µM
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| Treatment protocol |
After 0 h, 0.5 h, 4 h, 9 h and 24 h, cells corresponding to an OD600 of 3.5 in 1 l were harvested by centrifugation at 4 °C, 5000 x g and washed once in 20 ml CGXII (without MOPS). Subsequently, the cell pellet was resuspended in 20 ml CGXII (without MOPS) containing 1 % (v/v) formaldehyde to crosslink the regulator protein to the DNA. After incubation for 20 min at RT, the cross linking was stopped by addition of glycine (125 mM), followed by an additional 5 minutes of incubation. After that, the cells were washed three times in buffer A (100 mM Tris-HCl, 1 mM EDTA, pH=8.0) and the pellets stored overnight at -80 °C.
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| Growth protocol |
A preculture (C. glutamicum::hrrA-C-twinstrep) was inoculated in liquid BHI medium from a fresh BHI agar plate and incubated for 8-10 h at 30°C in a rotary shaker. After that, cells were transferred into a second preculture in CGXII medium containing 2 % (w/v) glucose and 0 µM FeSO4 to starve the cells from iron protocatechuic acid (PCA), which was added to the medium, allowed the uptake of trace amounts of iron. From an overnight culture, the main cultures were inoculated (CGXII medium containing 4µM heme) and harvested 0h, 0.5h, 2h, 4h, 9h or 24h after inoculation.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For cell disruption, the pellet was resuspended buffer A containing “cOmplete” protease inhibitor cocktail (Roche, Germany) and passed through a French pressure cell (SLM Ainco, Spectronic Instruments, Rochester, NY) five times at 172 MPa. The DNA was fragmented to ~500 bp by sonication (Branson Sonifier 250, Branson Ultrasonics Corporation, Connecticut, USA) and the supernatant was collected after ultra-centrifugation (150.000 x g, 4 °C, 1 h). The DNA-bound by the twin-Strep tagged HrrA protein was purified using Strep-Tactin XT Superflow column material (IBA Lifesciences, Göttingen, Germany) according to the supplier’s manual (applying the gravity flow protocol, 1.5 ml column volume). Washing of the column was performed with buffer W (100 mM Tris-HCl, 1 mM EDTA, 250 mM NaCl, pH 8,0) and the tagged protein was eluted with buffer E (100 mM Tris-HCl, 1 mM EDTA, 250 mM NaCl, pH 8.0, added 50 mM D-Biotin). After purification, 1 % (w/v) SDS was added to the elution fractions and the samples were incubated overnight at 65°C. For the digestion of protein, 400 µg/ml Proteinase K (AppliChem GmbH, Darmstadt, Germany) was added and incubated for 3 h at 55 °C. Subsequently, the DNA was purified as following: Roti-Phenol/Chloroform/Isoamyl alcohol (Carl Roth GmbH, Karlsruhe, Germany) was added to the samples in a 1:1 ratio and the organic phase was separated using Phase Lock Gel (PLG) tubes (VWR International GmbH, Darmstadt, Germany) according to the supplier’s manual. Afterwards, the DNA was precipitated by adding ice cold ethanol (to a conc. of 70 %) and centrifugation at 16.000 x g, 4 °C for 10 min. The DNA was washed with ice cold 70 % ethanol, then dried for 3 h at 50 °C and eluted in dH2O.
The resulting DNA was used for library preparation and indexing using the TruSeq DNA PCR-free sample preparation kit (Illumina, California, USA) according to the manufacturer’s instructions, only skipping fragmentation of the DNA. The resulting libraries were quantified using the KAPA library quant kit (Peqlab, Bonn, Germany) and normalized for pooling.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina MiSeq |
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| Data processing |
Data analysis and base calling were accomplished with the Illumina (MiSeq) instrument software and stored as fastq output files
The sequencing data obtained for each sample were imported into CLC Genomics Workbench (Version 9, Qiagen Aarhus A/S) for trimming and base quality filtering
The output was mapped to accession NC_003450.3 as C. glutamicum reference genome
Genomic coverage was convoluted with second order Gaussian kernel
The convolution profile was scanned in order to find points were first derivative changes its sign from positive to negative. This Points were considered as peaks and a score for each peak was set to the convolution value at he corressponding point
We fit the Gaussian curve to the distribution of peak scores (via optimize.fit function from SciPy packageand set a scopeak re thresholds equal mean + 4 sigmas of the fitted distribution
Genome_build: NC_003450.3
Supplementary_files_format_and_content: Processed data are in standard bed format (6 fields) format representing genomic positions of the peaks. Please note, that reported peaks are unstranded, even though strand is set to ‘+’ for the reported peaks
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| Submission date |
Nov 27, 2019 |
| Last update date |
Dec 01, 2019 |
| Contact name |
Andrei Filipchyk |
| E-mail(s) |
[email protected]
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| Phone |
+49 2461 61 2544
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| Organization name |
FZ-Juelich
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| Department |
IBG-1
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| Lab |
Frunzke AG
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| Street address |
Wilhelm-Johnen-Straße
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| City |
Juelich |
| ZIP/Postal code |
52428 |
| Country |
Germany |
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| Platform ID |
GPL25746 |
| Series (1) |
| GSE121962 |
ChAP-Seq analysis of HrrA in C. glutamicum |
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| Relations |
| BioSample |
SAMN13413417 |
| SRA |
SRX7229027 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4195575_time9.bed.gz |
4.3 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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