Computational protocol: Histone H1 Limits DNA Methylation in Neurospora crassa

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Protocol publication

[…] For chromatin immunoprecipitation (ChIP) experiments, 5 × 106 conidia/ml were inoculated into 50 ml of VMM and incubated at 32° for 5 hr. Cells were harvested and ChIP was performed as previously described () using anti-FLAG antibodies (cat # F1804; Sigma-Aldrich) or antibodies to the unphosphorylated C-terminal repeat of RNA polymerase II (8WG16; cat# MMS-126R; Covance). Two biological replicates were performed for each experiment. Libraries were prepared using the TruSeq ChIP sample prep kit (Illumina, cat # IP-202-1012) according to the manufacturer’s instructions with the following modification. Library amplification was performed using only four cycles of PCR to reduce biased enrichment of GC-rich DNA (). Libraries were sequenced at the University of Georgia Genomics Facility on an Illumina NextSeq 500 instrument. Reads were aligned to version 12 of the N. crassa genome (Refseq Accession # GCF_000182925.2; ) using the Burrows-Wheeler Aligner (BWA version 0.7.10) (). To determine if H1-3XFLAG was enriched over background, coverage was normalized to mitochondrial DNA as follows. We used BEDtools (version 2.25.0) ‘coverage’ to calculate read coverage for 1000 bp windows across the genome (). We then used BEDtools ‘map’ to calculate the median coverage for mitochondrial DNA. The coverage for each 1000 bp window was then divided by the median coverage for mitochondrial DNA. As a positive control, data from a previously published ChIP experiment for methylated lysine-9 of H3 was analyzed (Accession #SRX550120; ).The Hypergeometric Optimization of the Motif EnRichment (HOMER version 4.7.2) software package () was used to generate metaplots and heatmaps of enrichment data (annotatePeaks.pl module; using the -hist and -ghist option, respectively). We first created a custom HOMER genome annotation for Neurospora using a fasta file and a GTF file (Supplemental Material, File S1) containing the version 12 genome assemblies and annotations, respectively (). All plots were centered on transcriptional start sites or transcriptional termination sites, and a window size of 10 bp was specified for all histograms (-hist 10). HOMER was also used to construct metaplots of expression-ranked gene groups using the -list option. Genes were assigned into expression-ranked groups by expression level determined by RNA-seq (see below). RPKM values for each quintile group were: Q1 (12927.9–36.8); Q2 (36.8–13.8); Q3 (13.8–4.9); Q4 (4.9–0.33); Q5 (0.33–0). Thus, most genes in expression group 5 were silent or rarely expressed. [...] For RNA-seq experiments, 5 × 106 conidia/ml were inoculated into 50 ml of VMM containing 2% glucose and grown for 5 hr at 32°. RNA isolation was performed as described (; ), and strand-specific RNA-seq libraries were prepared from 5 μg total RNA. Ribosomal RNAs were depleted using the yeast Ribo-zero kit (cat # MRZY1324 Epicentre) and RNA libraries were generated with the Illumina Stranded RNA-seq kit (cat # RS-122-2101). Reads were aligned to version 12 of the N. crassa genome sequence using TopHat () and expression levels (RPKM) were determined using Cufflinks (). […]

Pipeline specifications

Software tools TopHat, Cufflinks
Application RNA-seq analysis
Organisms Neurospora crassa