|
| Status |
Public on May 02, 2020 |
| Title |
AflR_ChIPSeq |
| Sample type |
SRA |
| |
|
| Source name |
chromosome
|
| Organism |
Aspergillus flavus |
| Characteristics |
medium: PDB
incubation: 28℃ for 24 h
chip antibody: AflR (multiclonal antibody of AfAflR was produced in rabbits by Abcam (Shanghai, China); goat anti-rabbit antibody (anti-AfAflR) purchased from Abmart (Shanghai, China))
|
| Treatment protocol |
The cross-linking, DNA sonication, and chromatin immunoprecipitation were performed according to the protocols of Chung et al. Briefly, the chromatin was extracted and sonicated (Branson sonifier, Danbury, CT, USA) at half-maximal power over ten 10-sec pulses with chilling on ice for 2 min after each pulse. An aliquot of the chromatin solution (1/10 of the total volume) was used as input DNA to determine the DNA fragment sizes. The average sizes of the resultant DNA fragments were ~0.2–1.5 kb. The remaining chromatin solution was divided into two parts: one was incubated with the addition of 10 μl of the antibodies (anti-AfAflR), and the other was incubated without antibodies (mock). Immunoprecipitated DNA was used for sequencing. Millipore Chromatin Immunoprecipitation Assay Kit (17-295, EMD Millipore Corporation, Temecula, CA, USA) was used in ChIP experiments.
|
| Growth protocol |
A. flavus NRRL3357 was grown in 200 mL (1 × 10e6 spores/mL) of potato dextrose broth (PDB) in 500 mL shaking flasks at 28 °C for 24 h. Three replicate cultures were prepared. The cultures were centrifuged and transferred to a cross-linking solution for ChIP experiments.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
ChIP-sequencing was accomplished on the Illumina HiSeq 2500 with the ChIP-seq libraries.
Reads were trimmed and cleaned of Illumina adaptors using Trimmomatic and aligned to the A. flavus NRRL3357 genome using bowtie2-2.1.0. The genome and annotations of A. flavus NRRL3357 were downloaded from NCBI (The National Center for Biotechnology Information). Reads that aligned concordantly were used for peak calling.
The resulting bam files were used as an input for peak calling by Model-based Analysis for ChIP Sequencing (MACS2) version 2.0.10.20131216. Peak calling was done with the ChIP-seq samples and input control samples using a False Discovery Rate (FDR) cutoff of 0.05.
The topGO R package was used for functional enrichment and gene ontology (GO) analysis as described previously. The p-value cutoff was set at 0.05 for GO analyses.
The online motif finding program Multiple EM for Motif Elicitation (MEME, Version 5.1.1) was used to predict AfAflR-binding motifs within ChIP-seq peaks. The sequences of the 200 bp centered on each of the peaks were uploaded into MEME.
Genome_build: JCBI-AFL1-V2.0
Supplementary_files_format_and_content: wig files were generated using the Samtools.
|
| |
|
| Submission date |
May 01, 2020 |
| Last update date |
May 28, 2020 |
| Contact name |
Qing Kong |
| E-mail(s) |
[email protected]
|
| Phone |
+86 13573865136
|
| Organization name |
Ocean University of China
|
| Department |
Food Science and Engineering
|
| Street address |
Yushan Road 5
|
| City |
Qingdao |
| State/province |
Shandong |
| ZIP/Postal code |
266003 |
| Country |
China |
| |
|
| Platform ID |
GPL23153 |
| Series (1) |
| GSE149696 |
Identification of AflR binding sites in the genome of Aspergillus flavus by ChIP-seq |
|
| Relations |
| BioSample |
SAMN14790184 |
| SRA |
SRX8408708 |
