Computational protocol: Centromeres of the Yeast Komagataella phaffii (Pichia pastoris) Have a Simple Inverted-Repeat Structure

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Protocol publication

[…] Chromatin immunoprecipitation followed . Yeast cells were cultured in 200 ml YPD to log phase and then crosslinked with 1% formaldehyde at room temperature. Crosslinking was stopped by addition of 2.5 M glycine. Crosslinked cells were washed with TBS (100 mM Tris–HCl, pH 7.5, 150 mM NaCl) and resuspended in FA lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% v/v Triton X-100, 0.1% w/v sodium deoxycholate, 0.1% w/v SDS) containing 1 mM PMSF. Cells were lysed with glass beads and chromatin was fragmented by sonication with a Bioruptor Standard (Diagenode). Immunoprecipitation of chromatin fragments was performed with EZview Red Anti-HA Affinity Gel (Sigma–Aldrich) or mouse IgG1 (Cell Signaling Technology) as isotype control. After washes, bound DNA was eluted with HA peptide (Sigma–Aldrich), crosslinks were reversed, and the samples were purified by phenol–chloroform extraction before sequencing. Unpaired Illumina ChIP-seq reads (51 bp) were mapped to the 2011 version of the K. phaffii CBS7435 genome () including the mitochondrial genome, using BWA v0.7.9a-r786 (aln/samse algorithm) with default parameters (). Samtools v0.1.12a (r862) was used to create sorted and indexed BAM files of the results. Bedtools v2.19.0 was used to create genome coverage Bedgraph files to produce and . […]

Pipeline specifications

Software tools BWA, SAMtools, BEDTools
Application ChIP-seq analysis
Organisms Saccharomyces cerevisiae, Komagataella phaffii, Komagataella pastoris, Candida albicans, Schizosaccharomyces pombe