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Pipeline publication

[…] epared from ∼5 ng of ChIP DNA using the TruSeq ChIP Library Prep Kit (Illumina). Libraries were quantified on a high sensitivity DNA chip (Agilent)., Libraries were sequenced on Illumina MiSeq, HiSeq 2,000 and HiSeq 3,000 systems for collecting 50 base-pair single-end or 150 base-pair paired-end reads to high sequencing depth (). Only Read 1 of 150 base-pair paired-end reads were used and further trimmed to 50 base pair. Uniquely mapped 50 base-pair single-end reads were used for ChIP-seq analyses., Next generation sequencing (NGS) reads were mapped to Arabidopsis thaliana reference transcriptome TAIR10 ver. 24, with ribosomal RNA regions (2:3471–9557; 3:14,197,350–14,203,988) masked, using TopHat 2.0.13 (no-mixed alignments; up to 20 secondary alignments; no novel junctions). Counts of NGS reads covering transcripts were computed using the function summarizeOverlaps in R. Expressed genes were defined as those having the value of FPKM>1 at least at one time point. Read counts were submitted to differential gene expression analysis in Deseq2 (default parameters, FDR<0.05). Regularized logarithms of read count computed by Deseq2, denoted by rlog, were used for the analysis of relationships between gene expression level and histone modifications signal., NGS reads were mapped to Arabidopsis thaliana reference genome TAIR10 ver. 24 using Bowtie2 2.2.4 (minimum alignment score −12.5; uniquely mapped reads only). The sets of aligned H3K27me3 and H3 reads were down-sampled to minimum number of reads, respectively, for peak-calling from the same sequencing depth in all samples. Significant enrichment regions in H3K4me3 and H3K27me3 samples, relative to H3 control samples, were identified using MACS2 2.1.0 (default settings for H3K4me3—FDR<0.05; broad-peaks mode for H3K27me3—FDR<0.1). Regions with signal level measured by FPKM(ChIP)/FPKM(H3 control) differing between biological replications by more than 2 s.d. were filtered out. Consensus regions found as a sum of regions from two replications were taken as histone modifica […]

Pipeline specifications

Software tools TopHat, DESeq2, Bowtie2
Organisms Arabidopsis thaliana