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| Status |
Public on Nov 30, 2019 |
| Title |
A.pp 4074 NarP DAP-Seq |
| Sample type |
SRA |
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| Source name |
Actinobacillus pleuropneumoniae 4074 cells
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| Organism |
Actinobacillus pleuropneumoniae |
| Characteristics |
strain: 4074
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| Growth protocol |
A. pleuropneumoniae (strain 4074) was cultured at 37 °C in Tryptic Soy Broth (TSB) or on Tryptic Soy Agar plates (TSA, ) supplemented with NAD at 10 mg/ml.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The genomic DNA of A. pleuropneumoniae was prepared using a DNeasy Blood & Tissue Kit (Qiagen).
1 μg genomic DNA was sheared to an average length of 300-500 bp by ultra-sonication for 16 min at 20% amplitude with 2 seconds on and 8 seconds off on ice. The size of the sheared genomic DNA was checked using 1 % agarose gel. The sheared DNA fragments were treated with Klenow enzyme to generate blunt ends, phosphorylated and 3’-end adenylated followed by ligation with paired-end adapters. The fragments with adapters on both ends were enriched by a ten-cycle PCR and purified using a QIAquick PCR Purification Kit. The adapter-containing DNA fragments were then subject to DNA affinity purification. Briefly, the DNA fragments were mixed with the purified his-tagged NarP in the reaction buffer (10 mM Tris HCl, pH 7.5, 1 mM DTT, 5 mM MgCl2, 50 mM acetyl phosphate and 25% glycerol) in which acetyl phosphate was used to activate NarP. The mixture was incubated at 25 °C for 30 minutes and then incubated with 30 μl of Ni-NTA resin that had been equilibrated in binding buffer 2 (10 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 50 mM KCl, and 25% glycerol) for 30 min. The resin was washed three times with binding buffer 2 and the protein was eluted with elution buffer (20 mM sodium phosphate buffer pH 8, 500 mM NaCl, and 500 mM imidazole). The DNA bound to NarP was recycled using a Qiaquick PCR purification kit.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
454 GS |
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| Description |
The sheared genomic DNA bound with activated NarP.
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| Data processing |
Library strategy: DAP-seq
A total of 1022667 reads after trimming were aligned to the genome of the A. pleuropneumoniae 4074 strain using Bowtie2
Then it was analyzed using MACS2 (version 1.4.2 20120305) with defaulted settings to call peaks.
Potential promoter regions (upstream regions of any ORFs) fell in the peaks were taken as the potential targets that NarP bound.
Genome_build: ASM17849v1 [ADOD00000000.1]
Supplementary_files_format_and_content: bed format, it contains the locations for every peak, and the summit height of fragment pileup.
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| Submission date |
Nov 30, 2017 |
| Last update date |
Nov 30, 2019 |
| Contact name |
Lu Li |
| E-mail(s) |
sakura.tree@163.com, xuzf@mail.hzau.edu.cn, xuzhuofei@sohu.com, kobe2071@tom.com
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| Organization name |
Huazhong Agricultural University
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| Street address |
Shizishan Street 1
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| City |
Wuhan |
| State/province |
Hubei |
| ZIP/Postal code |
430070 |
| Country |
China |
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| Platform ID |
GPL24322 |
| Series (1) |
| GSE107526 |
Identification of the genes regulated by the two-component system response regulator NarP of Actinobacillus pleuropneumoniae by DNA-affinity-purified (DAP) sequencing |
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| Relations |
| BioSample |
SAMN08113336 |
| SRA |
SRX3432971 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2870527_A._pleuropneumoniae_NarP.sam.bam.srt_macs_p05_summits.bed.gz |
698 b |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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