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| Status |
Public on Jul 17, 2018 |
| Title |
Untreated Rep 3 -A-IP |
| Sample type |
SRA |
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| Source name |
Un_IN_3
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| Organism |
Escherichia coli |
| Characteristics |
strain: DY330
tagged gyrase subunit: GyrA-SPA
musgs presence: -
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| Treatment protocol |
When reaching the optical density indicated (OD600=0.6-0.8) the culture was bisected and DNA gyrase poison (ciprofloxacin, oxolinic acid or microcin B17) was added to the first half (+A samples), while second served as a control (-A samples).
Cultures (+A and -A) were incubated at 32°C with shaking for additional 15 min, then cells were pelleted by centrifugation at 10°C (4500xg) and resuspended in 10 ml of TES buffer (10 mM Tris-Cl pH7.5, 1 mM EDTA, 250 mM NaCl). Washing procedure was repeated twice to remove culture liquid and excess of gyrase poison.
Washed pellets were resuspended in 1 ml of TESS buffer (10 mM Tris-Cl pH7.5, 1 mM EDTA, 250 mM NaCl, 0.02% SDS, 0.2% Tween-20) with addition of proteases inhibitors cocktail (cOmplete ultra EDTA free, Roche) and RNAse A (Thermo Scientific). Resulting suspensions were sonicated with parameters optimized to obtain DNA fragments between 200 and 700 bp (SONOPULS HD 3100). Lysates were diluted with 1 ml of TES buffer and 100 μl of ANTI-FLAG® M2 affinity gel (Sigma-Aldrich) was added. Immunoprecipitation was performed for 1.5-2 hours at room temperature with moderate mixing, then affinity gel was washed 4 times by repeating steps of centrifugation (1.5 minute, 1000xg at room temperature) and resuspention (x2 with 1 ml of TESS buffer, x1 with 1 ml of TES buffer, x1 with 1 ml of TE buffer).
Lysates were diluted with 1 ml of TES buffer and 100 μl of ANTI-FLAG® M2 affinity gel (Sigma-Aldrich) was added. Immunoprecipitation was performed for 1.5-2 hours at room temperature with moderate mixing, then affinity gel was washed 4 times by repeating steps of centrifugation (1.5 minute, 1000xg at room temperature) and resuspention (x2 with 1 ml of TESS buffer, x1 with 1 ml of TES buffer, x1 with 1 ml of TE buffer).
For proteolysis, affinity gel obtained after the last centrifugation step was diluted with TES buffer up to final volume 200 μl, proteinase K (Sigma-Aldrich) was added (0.5 mg/ml) and samples were incubated at 55°C for at least 3 hours. After this step samples were centrifuged (2 minutes, 2000xg at room temperature) and supernatants were collected for DNA extraction.
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| Growth protocol |
1 ml of overnight culture was prepared for E. coli DY 330 GyrA-SPA or E. coli DY 330 GyrA-SPA MuSGS by seeding 2YT medium supplemented with antibiotics (kanamycin 50 µg/ml for DY 330 GyrA-SPA and kanamycin 50 µg/ml, chloramphenicol 15 µg/ml for DY 330 GyrA-SPA MuSGS) with cells from one isolate colony.
Starter was cultivated at 32°C with shaking (180 rpm), then was inoculated into 100 ml of 2YT without antibiotics and cultivation was proceeded under the same conditions until culture reaching mid-logphase (OD600=0.6-0.8).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted from resulting supernatant with phenol/chlorophorm method followed by ethanol precipitation. Mock controls (-IP) were made both for +A and for -A: 100 μl aliquots of lysates obtained after sonication were deproteinized and DNA was purified as described earlier. The procedure described gives a quartet of samples (+A+IP, +A-IP, -A+IP, -A-IP), where +A-IP, -A+IP, and -A-IP serves as controls for gyrase poison action and immunoprecipitation.
Sequencing libraries were prepared with Accel NGS 1S kit (Swift Bioscience) in accordanse with manufacturer’s protocol.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Untreated cells
Cell culture
Total DNA (mock DNA)
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| Data processing |
Raw reads were aligned to the E. coli w3110 MuSGS genome (containing a strong gyrase binding site from bacteriophage Mu) with BWA MEM (default settings)
Resulting SAM files were processed with custom script (SAM_to_coverage_and_N5E_N3E.py, github: https://github.com/sutormin94/Gyrase_Topo-seq), giving coverage depth of the genome and N3E values (number of DNA fragments 3'-ends) for every position
Gyrase Cleavage Sites (GCSs) were called using custom script (GCSs_calling.py, github: https://github.com/sutormin94/Gyrase_Topo-seq) that takes 4 files (tetrade) as input: +A+IP, +A-IP, -A+IP, -A-IP
Genome_build: Escherichia coli W3110 MuSGS (on the basis of NC_007779.1). FASTA and GFF are included on series record.
Supplementary_files_format_and_content: tar archives contain the following:
text wig files contain N3E values (number of DNA fragments 3' ends for each position of the genome)
text wig files contain coverage depth data
tab-delimited text files with GCSs coordinates, N3E values ("height of the peak") and GCSs scores (score of the sequence under the GCS obtained after scanning the genome with DNA-gyrase binding motif)
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| Submission date |
Jul 16, 2018 |
| Last update date |
Jul 17, 2018 |
| Contact name |
Dmitry Sutormin |
| E-mail(s) |
sutormin94@gmail.com
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| Phone |
+79154072592
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| Organization name |
Skolkovo Institute of Science and Technology
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| Department |
Life Sciences
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| Street address |
Nobelya Ulitsa 3
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| City |
Moscow |
| ZIP/Postal code |
121205 |
| Country |
Russia |
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| Platform ID |
GPL21222 |
| Series (1) |
| GSE117186 |
Topo-Seq application for topoisomerases binding sites identification with a single-nucleotide resolution |
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| Relations |
| BioSample |
SAMN09666208 |
| SRA |
SRX4396043 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3273137_Un_IN_3.tar.gz |
5.6 Mb |
(ftp)(http) |
TAR |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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