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Sample GSM4956738

Query DataSets for GSM4956738

Status

Public on Dec 05, 2020

Title

18DAP WT embryo ZmAGO4 ChIP-seq input Rep1

Sample type

SRA

Source name

Maize developing seed

Organism

Zea mays

Characteristics

strain: W22
tissue: developing embryo
genotype: WT
age: 18 DAP
chip antibody: none (input)

Treatment protocol

No special treatment to all of the biological materials.

Growth protocol

Maize plants were grown in the field at Beijing in the summer of 2017

Extracted molecule

genomic DNA

Extraction protocol

18 DAP embryo chromatin was isolated as previously described (Yang et a;.2016).Briefly, formaldehyde-fixed samples were ground to fine powder in liquid nitrogen. Nuclei pellets were suspended in a buffer containing 0.25 M sucrose, 10 mM Tris-HCl, pH 8, 10 mM MgCl2, 1% Triton X-100, 5 mM β-mercaptoethanol, 0.1 mM PMSF, and protease inhibitors (one mini tablet per milliliter; Roche). The suspensions were transferred to microfuge tubes and centrifuged at 12,000g for 10 min. The pellets were suspended in 1.7 M sucrose, 10 mM Tris-HCl, pH 8, 2 mM MgCl2, 0.15% Triton X-100, 5 mM β-mercaptoethanol, 0.1 mM PMSF, and protease inhibitors (one tablet in 30 mL solution; Roche), and centrifuged through a layer of the same buffer in microfuge tubes. The nuclear pellets were lysed in a buffer containing 50 mM Tris-HCl, pH 8, 10 mM EDTA, 1% SDS, and protease inhibitors (one tablet in 30 mL solution; Roche). The lysed nuclei were sonicated with a Bioruptor (UCD-200) in a water bath at 4°C for 12 cycles with 30s on and 30s off.Immunoprecipitation was performed using about 10 µg of chromatin, following a previously described protocol (Locatelli et al., 2009). Typically, 10 μL of affinity-purified antibody were used for immunoprecipitation. The precipitated DNA was dissolved in 50 μL 10 mM Tris-HCl, pH 7.5, 1 mM EDTA and treated with RNase (DNase-free).
Antibody pulled-down DNA was subject to regular DNA library preparation procedure with NEBNext Ultra II DNA library Prep Kit (New England BioLabs).

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2500

Description

ZmAGO4_ChIP-seq_WT_Rep1_peak

Data processing

For ChIP-seq, low quality reads and potential adaptor sequence was removed using Trim_galore (https://github.com/FelixKrueger/TrimGalore). Clean reads were then mapped to the reference B73 genome (Jiao et.al 2017) (release AGPV4) with Bowtie2 (-no-unal -I 0 -X 800 -no-discordant). Mapped reads with MAPQ>=5 were retained. Potential PCR duplicates was removed by MarkDuplicates V2.17.10 from Picard tools (https://github.com/broadinstitute/picard).
ChIP-seq peaks were called using MACS2 (v 2.1.2) with the following setting: -g 2.3e+9 -B -SPMR -q 0.01.
For mRNA-seq, low quality reads and adaptor sequence were trimmed using Trim_galore. Clean reads were subsequently mapped to maize reference genome (B73_RefGen_v4.43. gff3) with Tophat-2.0.10. Expression level of genes was calculated in FPKM (fragment per kilobase per million) using Cufflinks-2.2.1 packages.
Genome_build: B73_RefGen_v4
Supplementary_files_format_and_content: peak text files, bedGraph, FPKM for RNA-seq

Submission date

Dec 04, 2020

Last update date

Dec 05, 2020

Contact name

Jincheng Long

E-mail(s)

longjincheng1990@163.com

Phone

+8613001298733

Organization name

China Agricultural University