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Sample GSM2870527 Query DataSets for GSM2870527
Status Public on Nov 30, 2019
Title A.pp 4074 NarP DAP-Seq
Sample type SRA
Source name Actinobacillus pleuropneumoniae 4074 cells
Organism Actinobacillus pleuropneumoniae
Characteristics strain: 4074
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.
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.
Library strategy OTHER
Library source genomic
Library selection other
Instrument model 454 GS
Description The sheared genomic DNA bound with activated NarP.
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.
Submission date Nov 30, 2017
Last update date Nov 30, 2019
Contact name Lu Li
Organization name Huazhong Agricultural University
Street address Shizishan Street 1
City Wuhan
State/province Hubei
ZIP/Postal code 430070
Country China
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
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 SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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