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Sample GSM6153720

Query DataSets for GSM6153720

Status

Public on Jun 29, 2022

Title

ChIP-Seq of Fusarium graminearum PH-1:FgPacC30-GFP, input 2

Sample type

SRA

Source name

Mycelium

Organism

Fusarium graminearum PH-1

Characteristics

tissue: Mycelium
chip antibody: none
genotype: deletion mutant FgPacC
infection: NO
treatment: ChIP experiments using Day 2 of fresh mycelium

Extracted molecule

genomic DNA

Extraction protocol

fresh mycelia of each sample were cross-linked with 1% formaldehyde for 10 min. Fixation was stopped with 125 mM glycine for 5 min. Subsequently, fixed culture samples were grounded in liquid nitrogen, and re-suspended in 10 ml of nuclear extraction buffer I (0.4 M sucrose, 10 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 5mM β-mercaptoethanol, 0.1 mM PMSF, 1× protease inhibitor). DNA was sheared into 200~500 bp fragments with 30 s on and 6 min off by using a Covaris E220 DNA Sonicator (Covaris, Woburn, MA). After centrifugation, the pellets were suspended in 4 ml of nuclear extraction buffer II (1.7 M sucrose, 10 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 1% Triton X-100, 5 mM β-mercaptoethanol and 1×protease inhibitor). Re-suspended samples were then centrifuged at 13,000 g for 15 min at 4 °C. The resulting pellets were re-suspended in 300 μL of nuclear lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS and 1× protease inhibitor). Samples were then sonicated by two pulses of 30 s sonication and 1 min rest. After centrifugation at 4000 g for 5 min at 4°C, the supernatant was transformed into a clear tube, and was diluted with 10×ChIP dilution buffer (1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.0 and 167 mM NaCl). Immunoprecipitation was conducted using the monoclonal anti-GFP (ab290, Abcam, Cambridge, UK) antibody, anti-H3K18ac (39755, Active Motif Inc., Carlsbad, USA) antibody, or anti-H2BK11ac (ab240613, Abcam, Cambridge, UK) together with the protein A agarose beads (sc-2003, Santa Cruz, CA, USA). The beads were subsequently washed by low salt wash buffer, high salt wash buffer, LiCl wash buffer, and TE buffer. After washing, eluting, reversing the crosslinking, and removing all proteins, the resulting pellets were re-suspended in 50 μL of distilled water.
End repair and adapter ligation:Fill the ends of IP/Input DNA followed by 5' phosphorylation. A 3' sticky-end with an overhanging A is formed, which could ligate with an overhanging T at the 3' end of the bubble adapter; PCR amplification: PCR amplify the ligated products with specific primers; Single-strand separation and circularization: Separate PCR products into single-strands by thermal denaturation. Then circularize the single-stranded DNA through a bridge primer to obtain single-stranded circular DNA library; Sequencing: Amplified products DNA Nanoballs (DNBs) are used for sequencing

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 4000

Data processing

For ChIP-seq analysis, 50 bp single-end reads of ChIP-seq were obtained, and quality was controlled using SOAPnuke(v2.1.7). The parameters were as follows: remove reads containing adapters or more than 1% of unknown nucleotides (N); delete the read if there are more than 40% bases having a quality value lower than 20.
Clean reads were mapped to the Fg reference genome by Bowtie2 (version 2.4.5) with default parameters.
The visualization of the average read coverage over 2 kb upstream and downstream of the TSS was performed by Deeptools.
BigWig files generated by Deeptools were used for visualization in Integrative Genome Viewer (version 2.8.9).
RPKM (Reads Per Kilobase per Million mapped reads) were calculated to normalized reads using bamCompare tool in Deeptools (version 2.4.1) with input DNA as a contorl.
Genome build: ASM24013v3 (reference genome of F. graminearum strain PH-1)
BigWig

Submission date

May 16, 2022

Last update date

Jun 29, 2022

Contact name

Yujie Wang

E-mail(s)

2019202005@njau.edu.cn

Organization name

Nanjing Agricultural University

Street address

No. 1, Weigang, Xuanwu District