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[…] Log-phase yeast cells were incubated at 18°C for 2 hours and then fixed for 30 minutes in 3% freshly made formaldehyde. The crosslinking reaction was stopped by the addition of 2.5 M Glycine to make a final concentration of 0.125 M. The cells were pelleted and washed with PBS (phosphate buffered saline) before resuspended in ChIP lysis buffer (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1% Triton X-100, 0.1% Deoxycholate supplemented with cOmplete protease inhibitors cocktail (Roche)). Ice cold glass beads were added and the mixtures were vigorously disrupted in a beadbeater. The lysates were collected and subjected to sonication to reduce chromatin size to 500–1000 base pairs. The cleared cell lysates were incubated with antibodies: H3K9me3 (Abcam), H3K9me2 (Abcam), and Flag (Sigma) over night at 4°C. Protein G beads were then added to isolate the antibodies and associated chromatin fragments. The beads were then washed with ChIP lysis buffer twice, ChIP lysis buffer containing 0.5 M NaCl, Wash buffer (10 mM Tris, pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% Deoxycholate, 1 mM EDTA), and TE (50 mM Tris pH 8.0, 1 mM EDTA). The bound chromatin fragments were eluted with TES (50 mM Tris pH 8.0, 1 mM EDTA, 1% SDS) and the crosslinking was reversed by incubating at 65°C overnight. The protein DNA mixture were then subjected to proteinase K treatment and phenol:chloroform extraction before the DNA was precipitated by ethanol.Quantitative real-time PCR (qPCR) was performed with Maxima SYBR Green qPCR Master Mix (Fermentas) in a StepOne Plus Real Time PCR System (Applied Biosystems). DNA serial dilutions were used as templates to generate a standard curve of amplification for each pair of primers, and the relative concentration of target sequence was calculated accordingly. An act1 fragment was used as a reference to calculate the enrichment of ChIP over WCE for each target sequence. A list of DNA oligos used is provided in .For ChIP-seq, DNA samples were prepared according to TruSeq ChIP sample preparation guide (Illumina) and sequenced on the Illumina HiSeq 2500 system by 100 bp paired-end sequencing. The raw reads were trimmed by Trimmomatic (v0.35) () to remove potential adapter contamination and regions with bad sequencing qualities. The trimmed reads were aligned to the S. pombe reference genome (Ensembl version: ASM294v2.29) by bwa (0.7.12-r1039) (http://bio-bwa.sourceforge.net/). The resulting sam files were further processed by Samtools (v1.2) (), picard-tools (v2.0.1) (http://broadinstitute.github.io/picard/) and GATK (v3.5) () for indexing, sorting, removing PCR duplicates, and local-realignment. The per-based mapping depth was calculated by bedtools (v2.25.0) () and the sliding window plots (window size = 100 bp, step size = 50 bp) were created by in-house Perl and R scripts. We also employed MACS (v1.4.2) () to contrast each ChIP sample versus WCE sample for peak calling. [...] X-ray diffraction data were collected at 100K at NE-CAT beamline 24-ID-C of Advanced Photon Source (APS) at Argonne National Laboratory and processed using program HKL2000 (). The structure was solved through molecular replacement using Phaser-MR in program PHENIX (). The structure refinement was carried out using PHENIX, and manual model building with Coot (). […]

Pipeline specifications

Software tools PHENIX, Coot
Applications Small-angle scattering, Protein structure analysis
Organisms Schizosaccharomyces pombe, Homo sapiens
Chemicals Methionine, Potassium, S-Adenosylmethionine