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Protocol publication

[…] lipore; 07-353), anti-H3K23Ac (Millipore; 07-355), anti-H3Ac (Millipore; 06-599) and anti-H4Ac (Millipore; 06-598)] together with Magnetic Protein G Beads (Promega, G747A) were added to the sonicated chromatin followed by incubation overnight to precipitate bound DNA fragments (2 μg antibody per each chip reaction for all the antibodies used in this paper). DNA was eluted and amplified by primers corresponding to genes of EIN3-R and EIN3-NR. Primers used in the paper are listed in ., Chromatin-immunoprecipitated DNA was sequenced using an Illumina HiSeq 2000 platform according to standard operating procedures. Single-end 51-bp reads were first mapped to the Arabidopsis genome (TAIR10) using Bowtie2 software (version 2.1.0) with default parameters. The quality control of Chip-seq was shown in . Duplicate reads were removed using SAMtools. For each histone modification in each condition, mapped reads were pooled across ChIP-seq replicates as described. Pooled reads were normalized as reads per kilobase per million mapped reads (RPKM) in windows of 50 bp using deep Tools, and then were visualized with the Integrative Genomics Viewer (IGV) (). In addition to validate our peak calling result by pooled peaks, we also did analysis using overlapped differential peaks, and the result is very similar to that called by using pooled reads. On the basis of a recent publication, we used pooled reads results in this paper. Peaks significantly enriched in ChIP-seq tags were identified by Model-based Analysis for ChIP-Seq (MACS2, version; parameters: --nomodel, -p 0.01). Differential peaks were identified using ‘MAnorm' method. For this method, the normalized M value (M=log2 (read density in C2H4 treated sample per read density in air treated sample) represents log2-transformed fold changes of enrichment intensities at each peak region. Thus, an absolute threshold value of M≥0.4 was used to select differentially enriched peaks. Genes within 2 kb of the peak regions were marked as associated genes. Biological functions of associated genes were assessed by agriGO., Proteins were resolved by SDS–PAGE and electroblotted onto a nitrocellulose membrane and probed with the indicated primary antibodies and then with secondary goat anti-rabbit (Bio-Rad 170-6515) or goat anti-mouse (Bio-Rad 170-6516) antibodies conjugated with horseradish peroxidase. The signals were detected by a chemiluminescence reaction using the SuperSignal kit (Pierce). Polyclonal anti-EIN2 antibodies were used at dilution of 1:4,000. Polyclonal anti-histone H3 (BioMol) was used at dilution of 1:5,000. Monoclonal anti-HA (Cell Signaling) was used at dilution of 1:5,000., Total RNA was extracted using a Qiagen Plant Total RNA Kit (Sigma) from 3-day-etiolated seedlings treated wit […]

Pipeline specifications

Software tools Bowtie2, SAMtools, IGV, MACS, MAnorm, agriGO
Organisms Arabidopsis thaliana