Computational protocol: Structure and function of human histone H3.Y nucleosome

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Protocol publication

[…] The H3.Y nucleosome was concentrated to 2.5 mg/ml. The crystals of the H3.Y nucleosome were obtained by the hanging drop vapor diffusion method, by mixing equal volumes of the sample and the reservoir solution (100 mM sodium acetate (pH 4.6), 0.14 M MnCl2, 12% 2-propanol, and 6% trimethylamine N-oxide dihydrate) at 20°C. Crystals were transferred into the cryoprotectant solution, containing 100 mM sodium acetate (pH 4.6), 0.14 M MnCl2, 30% PEG400 and 2% trehalose at 4°C and were flash cooled in a stream of N2 gas (100 K).The data set was collected at the BL-17A beamline in the Photon Factory (KEK) and the BL41XU beamline in SPring-8. Data set indexing and scaling were performed with the HKL2000 program (). The structure of the H3.Y nucleosome was solved by the molecular replacement method using the Phaser program (), with the human nucleosome structure (PDB ID: 3AV2) as the guide. All refinements of the model were performed using the PHENIX program (). The model was subjected to rigid body refinement in the initial refinement. After the rigid body refinement, iterative rounds of refinements, including the xyz coordinates, the real-space, the occupancies and the individual B-factors, were performed. Manual model building was performed using the COOT program (). The final structure showed no outliers in the Ramachandran plot, as evaluated with the MolProbity program (Supplementary Table S1) (). All structure figures were made using PyMOL (Schrödinger; [...] The mRNA-seq library was prepared using a TruSeq RNA-Seq sample preparation kit (Illumina K.K.; USA), and the samples were sequenced on an Illumina HiSeq1500 system. The gene expression levels (FPKM; Fragments per kilobase of exon per million mapped sequence reads) were estimated using the Tophat (version 2.0.8) and Cufflinks (version 2.0.1) programs with the default parameters (). The eleven expression level groups, named Zero, q0-10th, q10-20th … and q90-100th, were defined according to the FPKMs of the genes. The members of the Zero group have exactly FPKM = 0, and the others were divided into 10 groups with the deciles of all FPKMs; i.e. the genes were sorted by the FPKMs and assigned into 10 equal groups.The ChIP-seq library was prepared as described previously (). The samples were sequenced on an Illumina HiSeq1500 system. The sequenced reads were mapped onto the human genome (hg19) with the Bowtie program (version 2.2.2), using the default parameters, and the unique-hit reads were utilized for further analysis (see Supplementary Table S2 for read number statistics). The normalized ChIP-Seq signal intensities were calculated, as follows. To calculate the signal intensity of the ChIP-seq data, we first counted the mapped reads on a 1000 base-pair stretch with 10% overlapping windows (bins) throughout the human genome, and then the counts were normalized as Reads Per Kilobases Per Million reads (RPKMs) (). The input-normalized ChIP-Seq signal intensities were calculated as the RPKM differences between the ChIP and input DNA-control data (ChIP–input) on each bin. The ChIP-seq peaks of GFP-H3.Y were identified using MACS (version 2.0.10) () with the following parameters: callpeak–gsize hs–nomodel–broad–extsize 144–to-large–P value 1e-3. […]

Pipeline specifications

Software tools TopHat, Cufflinks, Bowtie
Application ChIP-seq analysis
Organisms Homo sapiens