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| Status |
Public on Jul 03, 2020 |
| Title |
GX Fis IP Bio-replicate 1 |
| Sample type |
SRA |
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| Source name |
lab strain
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| Organism |
Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344 |
| Characteristics |
strain: GX
growth phase: Early exponential
type: IP
chip antibody: mouse anti-c-Myc
bio-replicate number: 1
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| Treatment protocol |
Cells harvested at early exponential phase.
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| Growth protocol |
Overnight cultures were diluted to a starting A600 of 0.003 in 25 ml LB broth and grown to an A600 of 0.4.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were harvested by centrifugation at 3,220 x g for 10 min at room temperature and resuspended in 50 ml PBS. Formaldehyde was added dropwise to a final concentration of 1% (v/v) while continuously stirring the cells. After 10 min of crosslinking the reaction was quenched with cold glycine (2M) added dropwise to a final concentration of 0.125 M. Cells were stirred further for 5 min and then harvested by centrifugation at 3220 x g for 10 min at 4 °C. The pellet was suspended in 600 μl Lysis buffer (50 mM Tris-HCl pH 8.1, 10 mM EDTA pH 8.0, 1% (w/v) SDS, Roche protease inhibitor cocktail), incubated for 10 min on ice, prior to the addition of 1.2 ml IP Dilution Buffer (20 mM Tris-HCl pH 8.1, 150 mM NaCl, 2 mM EDTA pH 8.0, 1% (v/v) Triton X-100, 0.01% (w/v) SDS, Roche protease inhibitor cocktail) and transferred to a sonication tube. Chromatin was sheared to an average length of 500 bp by sonicating for 30 seconds with an amplitude of 10 µm, twelve times, with a 1 min incubation on ice between bursts using a Soniprep 150. Sheared chromatin was analysed by agarose gel electrophoresis and stored at -80°C. To pre-clear chromatin for input samples, 50 µl normal rabbit IgG (Millipore, Cork, Ireland) was added and samples were incubated for 1 h on a blood tube rotator SB1 at 4oC. Antibody was removed by the addition of 100 µl homogeneous Protein G-Agarose (Roche) and a further incubation for 3 h. The Protein G-Agarose was removed by centrifugation at 3,220 x g for 2 min and 200 µl was used as input. For all ChIP experiments a mock IP and an experimental IP were set up at the same time using 1,350 µl pre-cleared chromatin and 10 µg of normal mouse IgG (Millipore) and monoclonal mouse anti-c-Myc respectively. Samples were incubated at 4oC for 16 h on a blood tube rotator SB1. 100 µl homogeneous Protein G-Agarose was added to each and the samples were incubated for a further 3 h. Protein G-Agarose beads were pelleted by centrifugation at 5,200 x g, washed 4 times with high salt IP wash buffer (50 mM HEPES pH 7.9, 500 mM NaCl, 1 mM EDTA, 0.1% (w/v) SDS, 1% (v/v) Triton X-100, 0.1% (w/v) deoxycholate), and twice with TE pH 8.0. Protein-DNA complexes were eluted from the beads twice with IP elution buffer (100 mM NaHCO3, 1% (w/v) SDS). DNA was purified and recovered by performing a standard phenol‐chloroform extraction, followed by ethanol precipitation with 5 µg of glycogen (Invitrogen, Cat. no. 10814‐010). The DNA pellets of the IP samples were re‐suspended (by heating at 37°C) in 50 µl of sterile filtered water for experimental and mock IPs, and 100 µl for the Input DNA samples.
ChIP sequencing was performed by Vertis Biotechnologie AG using Illumina NextSeq 500 technology. The DNA samples were first fragmented with ultrasound (2 pulses of 30 sec at 4°C). After end-repair, TruSeq sequencing adapters were ligated to the DNA fragments. Finally, the DNA was PCR-amplified to about 10-20 ng/µl using a high fidelity DNA polymerase. Aliquots of the PCR amplified libraries were examined by capillary electrophoresis (Bioanalyzer, Agilent). For Illumina NextSeq sequencing, the samples were pooled in approximately equimolar amounts. The DNA pool was eluted in the size range of 250 – 550 bp from a preparative agarose gel. An aliquot of the size fractionated library pool was analyzed by capillary electrophoresis (Bioanalyzer, Agilent). The adapters were designed for TruSeq sequencing according to the instructions of Illumina. The shotgun library pool was sequenced on an Illumina NextSeq 500 system using 75 bp read length.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
MB_GX_IP_1
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| Data processing |
Alignment: bwa + samtools
Coverage calcuation (.bw files): deeptools
Peak calling: MACS2
Genome_build: NC_016810, NC_017718, NC_017719, NC_017720
Supplementary_files_format_and_content: narrow_peak_list_fdr_cutoff_annotated.xlsx: peak lists generated using MACS2 and roughly annotated using custom script; .bw: BigWig files for looking at coverage in a genome browser like IGB
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| Submission date |
Jun 10, 2020 |
| Last update date |
Jul 04, 2020 |
| Contact name |
Aalap Mogre |
| E-mail(s) |
aalap.mogre@gmail.com
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| Organization name |
Trinity College Dublin
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| Department |
Department of Microbiology
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| Lab |
Dorman Lab
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| Street address |
Moyne Institute of Preventitive Medicine
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| City |
Dublin |
| ZIP/Postal code |
D2 |
| Country |
Ireland |
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| Platform ID |
GPL20056 |
| Series (2) |
| GSE152225 |
Network rewiring: physiological consequences of reciprocally exchanging the physical locations and growth-phase-dependent expression patterns of the Salmonella fis and dps genes. [ChIP-Seq] |
| GSE152228 |
Network rewiring: physiological consequences of reciprocally exchanging the physical locations and growth-phase-dependent expression patterns of the Salmonella fis and dps genes. |
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| Relations |
| BioSample |
SAMN15203124 |
| SRA |
SRX8524896 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4609564_MB_GX_IP_1.sorted.bw |
3.0 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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