Computational protocol: EIN2-dependent regulation of acetylation of histone H3K14 and non-canonical histone H3K23 in ethylene signalling

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Protocol publication

[…] RNA from wild-type, ctr-1-1, and EIN2S645A-YFP-HA transgenic lines treated with 4 h ethylene gas or hydrocarbon-free air were isolated following the manufacturer's recommendation using the RNeasy Plant Kit (Qiagen). cDNA sequencing libraries were prepared according to the instructions included in the Illumina TruSeq v2 library preparation kit. Reads were mapped using TopHat, and analyzed using Cufflinks. Differentially expressed genes were identified by fragments per kilobase per million reads (FPKM) filter<0.1, requiring a twofold change comparing the indicated conditions with P≤0.05 after Benjamini–Hochberg correction. [...] 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. […]

Pipeline specifications

Software tools Bowtie2, SAMtools, IGV, MACS, MAnorm, agriGO
Databases TAIR
Application ChIP-seq analysis
Organisms Arabidopsis thaliana