Computational protocol: The octamer is the major form of CENP-A nucleosomes at human centromeres

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Protocol publication

[…] Paired-end ChIP-Seq reads were aligned to the human genome build hg19 with Bowtie2 version 2.0.0 using paired-end mode. Reads were aligned using a seed length of 50 bp and only the single best alignment per read with up to 2 mismatches was reported in the SAM file. The aligned mate pairs were joined in MATLAB using the ‘localalign’ function (to determine the overlapping region between the reads [requiring ≥95% overlap identity]) (). Duplicate read removal was carried out using the ‘rmdup’ command in SAMtools. To create nucleosome occupancy maps at neocentromeres, all joined reads were aligned to the neocentromere and the number of reads that align with 100% identity are plotted for each particular base pair along the neocentromere coordinate (). For analysis of α-satellite DNA, all joined reads were aligned to the dimerized α-satellite consensus sequence and those reads aligning with ≥ 60% identity were chosen for further analysis (). [...] Molecular models were generated using PDB ID 1KX5 and 1ZBB for the H3-containing particles and 3AN2 for CENP-A-containing particles. Models of tetrasomes and hemisomes with crossed DNA were generated using linker DNA from 1ZBB and minimized using CNS,. The model of the CENP-A nucleosome core particle was generated using DNA from 1KX5. The point in space of DNA crossing was determined as the shortest distance along the projection angle of the DNA between entry and exit sites. All molecular structure figures were generated using PyMOL ( […]

Pipeline specifications

Software tools Bowtie2, SAMtools
Application ChIP-seq analysis
Organisms Homo sapiens