The Involvement of Zyxin in DNA Binding, Gene Regulation, and Apoptosis within a Prostate Cancer Model.

Chromatin Immunoprecipitation sequencing (ChIP-seq) assays were performed using PC3M cells to identify the nuclear DNA targets of zyxin. The cells were crosslinked using formaldehyde, followed by cell lysis, chromosomal DNA fragmentation, and immunoprecipitation using anti-zyxin antibodies (Sigma# R98938) (or IgG as a control). After DNA extraction, library was prepared, next-generation sequencing was conducted on Illumina HiSeq 25000 (50bp SE, standard Illumina adapters) to identify the DNA targets of zyxin. Data analysis involved quality control of 60 million Illumina reads per sample, removal of low-quality reads, and elimination of adapter sequences. The reads were then mapped to the human genome (version hg38) using Bowtie2 (PMID 22388286). Peak calling was performed using Macs2 (PMID 22936215), and differential peaks were annotated using Genomation (PMID 25417204). For bioinformatics pipeline we used community driven and curated pipeline at https://nf-co.re/chipseq/2.0.0 . GRCh37 version of human genome model was used. We used Nextflow as task manager and Singularity images on High Performance Computer.