Contain code for MORC and TF project
small RNA-seq analysis
Small RNA-seq reads were downloaded from a previous paper. Adaptor sequence (TGGAATTCTCGG) was trimmed with trim_galore, and trimmed reads were mapped to the reference genome TAIR10 using Bowtie2 with only one unique hit and zero mismatches.
ATAC-seq analysis
ATAC-seq read adaptors were removed using trim_galore. The reads were then mapped to the Arabidopsis thaliana reference genome, TAIR10, using Bowtie2 (-X 2000 -m 1). Reads of chloroplast and mitochondrial DNA were filtered out and duplicate reads were removed using Samtools. ATAC-Seq open chromatin peaks of each replicate were called using MACS2 with parameters of -p 0.01 --nomodel --shift -100 --extsize 200. Consensus sets of chromatin peaks for all samples were merged by bedtools (v2.26.0) intersect allowing a distance of 10 base pairs. Following this, edgeR was used to define significant changes between peaks [Fold Change, (FC) > 2 and False Discovery Rate, (FDR) < 0.05]. ATAC-seq peak distributions were annotated using ChIPseeker. TF footprints were analyzed by TOBIAS with 572 plant TF motifs downloaded from JASPAR (http://jaspar.genereg.net/).
RNA-seq analysis
Cleaned short reads were aligned to the reference genome, TAIR10, by Bowtie2 (v2.1.0). Expression abundance was then calculated by RSEM using the default parameters. Heatmaps were visualized using the R package pheatmap. Differential expression analysis was conducted using edgeR. A threshold of p-value < 0.05 and Fold Change > 2 were used to decide whether there were any significant differences in expression between samples.
ChIP-seq analysis
ChIP-seq data was aligned to the TAIR10 reference genome with Bowtie2 (v2.1.0), only including uniquely mapped reads without any mismatches. Duplicated reads were removed by Samtools. ChIP-seq peaks were called by MACS2 (v2.1.1) and annotated using ChIPseeker [36]. Differential peaks were called by the bdgdiff function in MACS2 . ChIP-seq data metaplots were plotted by deeptools (v2.5.1). Correlation of MORC7 with ChIP-seq data was conducted with ChromHMM. H3K9ac, H3K27ac, H4K16ac, H3K4me1, H3K4me3, H3K36me2, H3K36me3, H3K9me2, H3K27me3, Pol II, and Pol V, as published previously, were included in this analysis (Additional file 6: Table S5). Motif enrichment analysis was performed with MEME (v5.0.5).
Whole-genome bisulfite sequencing (BS-seq) analysis
Previously published whole-genome bisulfite sequencing data for morc-mutants and wild type was reanalyzed. Briefly, Trim_galore (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) was used to trim adapters. BS-seq reads were aligned to the TAIR10 reference genome by BSMAP (v2.90), allowing two mismatches and one best hit (-v 2 -w 1). Reads with three or more consecutive CHH sites were considered to be unconverted reads and were filtered out. DNA methylation levels were defined as #C/ (#C + #T).