Nucleosome mapping

Semi-intact Yeast cell preparation – Semi-intact cells were prepared as previously described⁶⁰. Briefly, cells were grown at 30 °C in 300 ml YPD to =1 ×107 cells/ml. For each 250 ml of cells (10⁷cells/ml), semi- intact cells were prepared as follows. Cells were collected by centrifugation (700 × g, 7 min, RT), resuspended in 25 ml 100 mM Pipes, pH 9.4, 10 mM DTT, incubated with gentle agitation at 30 °C for 10 min, and collected by centrifugation (1000 × g, 5 min, RT). Cells were resuspended in 6 ml YP, 0.2% glucose, 50 mM KPO4, pH 7.5, 0.6 M sorbitol. 10 u zymolase was added, and the suspension was incubated with gentle shaking 30 °C for 30 min. Spheroplasting was monitored by light microscopy. Great care was taken not to overdigest cells to avoid lysis. Spheroplasts were collected by centrifugation at 1000 × g for 5 min at RT, re- suspended with a plastic pipette in 40 ml YE 1% glucose, 0.7 M sorbitol, and incubated with gentle shaking at 30 °C for 20 min. Spheroplasts were collected by centrifugation (1000 × g, 5 min, RT) and washed twice at 4 °C with cold permeabilization buffer (20 mM Pipes-KOH, pH 6.8, 150 mM K-Acetate, 2 mM Mg-Acetate, 0.4 M sorbitol. The final pellet was resuspended in 1 ml cold permeabilization buffer containing 10%(v/v) DMSO. 100 µl aliquots were placed in 1.5 ml microfuge tubes and frozen slowly above liquid N₂ and stored at −80 °C.

MNase-seq – 0.4 ×109 semi-intact cells were digested with micrococcal Nuclease (MNase), 1.5 unit at 37 °C for 30 min with 3 mM CaCl2. The reactions were stopped by addition of EDTA to a final concentration of 0.02 M and subsequently incubated with RNase A (0.1 mg) for 4 h at 37 °C and further treated with Proteinase K at 37 °C o/n. DNA was purified using phenol–chloroform extraction and concentrated by ethanol precipitation.

The percentage of mononucleosomal DNA fragments was examined by 2% agarose gels. Furthermore, the integrity and size distribution of digested fragments were determined using the microfluidics-based platform Bioanalyzer (Agilent) prior to library preparations following Illumina standard protocol. The short-insert paired-end libraries for MNase sequencing were prepared with PCR free protocol using KAPA Library Preparation kit (Roche). In short, 2.0 micrograms of Micrococcal nuclease (MNase) digested genomic DNA from S. cerevisiae was end-repaired, adenylated and ligated to Illumina platform compatible adapters with dual indexes (Integrated DNA Technologies). The adapter-modified end library was size selected and purified with AMPure XP beads (Agencourt, Beckman Coulter). The final libraries were quantified by Kapa Library Quantification Kit for Illumina platforms (Roche).

The libraries were sequenced using TruSeq SBS Kit v4-HS (Illumina), in paired-end mode with a read length of 2x76bp following the manufacturer’s protocol. Images analysis, base calling and quality scoring of the run were processed using the manufacturer’s software Real Time Analysis (1.18.66.3).

Nucleosome calling – MNase-seq paired-end reads were mapped to yeast genome (sacCer3, Apr. 2011) using Bowtie⁶¹ aligner, allowing a maximum of 2 mismatches and maximum insert size of 500 bp. Output BAM files were imported in R^62,58 and quality control was performed with htSeqTools package to remove PCR artifacts⁶³. Filtered reads were processed with nucleR package⁶⁴ as follows: mapped fragments were trimmed to 50 bp maintaining the original center and transformed to reads per million. Then, noise was filtered through Fast Fourier Transform, keeping 2% of the principal components, and peak calling was performed using the parameters: peak width 147 bp, peak detection threshold 35%, maximum overlap of 80 bp, dyad length 50 bp. Nucleosome calls were considered well-positioned when nucleR peak width score and height score were higher than 0.6 and 0.4, respectively, and fuzzy otherwise.

Nucleosome Dynamics – NucDyn R package⁴⁵ was used to find changes in nucleosome organization between control and methylation-induced samples. P-values quantifying the nucleosome change were obtained running NucDyn with the following parameters: maximum difference of 70, maximum length of 140, minimum number of reads to report a shift of 3, shifts threshold of 0.1, indels minimum number of reads to report evictions and inclusions (indels) of 3, indels threshold of 0.05.

Genomic Annotation – Data was annotated from the UCSC gene track that contains 6692 genes. We discarded genes that are described as “Putative” or “Dubious” and genes located in the mitochondrial chromosome. We used gene lengths to normalize methylation proportions, nucleosome coverages and CpG density partitioning each gene in 137 bins (each bin has on average 10 bp since the mean length of yeast genes is 1369 bp).