Computational protocol: The Promyelocytic Leukemia Zinc Finger Transcription Factor Is Critical for Human Endometrial Stromal Cell Decidualization

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Protocol publication

[…] Human ESCs were cultured in EPC media for 72 hours before PLZF and Input ChIP-Seq were performed by Active Motif, Inc. (Carlsbad, CA). Human ESCs derived from six subjects were pooled before being fixed in 4% formaldehyde, snap-frozen, and shipped on dry ice to Active Motif. A goat polyclonal anti-human PLZF antibody (sc-11146; Santa Cruz Biotechnology, Inc.) was used for the PLZF ChIP assay. Enriched DNA from the ChIP assay was amplified and DNA libraries were sequenced on an Illumina platform, followed by alignment of sequenced reads (24 million reads) to the human genome (GRCh37/hg19, February 2009). Peak calling was performed by applying a threshold of 18 (5 consecutive bins containing 0.18 aligns) before the resultant-called peaks were stored as Browser Extendable Data (BED) files. Due to the pronounced enrichment for TSS-proximal binding of PLZF by Cis-regulatory Element Annotation System (CEAS) analysis, genes associated with PLZF binding were called if the ChIP binding intervals were located within 950 bp from the TSS. Galaxy/Cistrome’s implementation of the Cis-regulatory Element Annotation System (CEAS) and Genomic Regions Enrichment of Annotations Tool (GREAT) tools were used for enrichment analyses on chromosomes and genomic annotations. Database for Annotation, Visualization, and Integrated Discovery Bioinformatics Resources (DAVID) v6.7 (http://david.abcc.ncifcrf.gov/)) and Gene Set Enrichment Analysis (GSEA) on Molecular Signatures Databases (MSigDB) collection (http://software.broadinstitute.org/gsea/index.jsp) analyses were performed on protein coding genes to identify enriched biological themes. Integration of PLZF ChIP-Seq data with RNA-Seq data sets was performed using a relational database system []. ChIP validation assays were performed on hESCs cultured in EPC media for seventy-two hours (Active Motif). A goat polyclonal anti-human PLZF antibody and a rabbit anti-human PGR (H-190) antibody (sc-7208, Santa Cruz Biotechnology, Inc.) were used to immunoprecipitate PLZF and PR proteins with associated bound DNA from fragmented chromatin respectively. Enriched DNA fragments from the PLZF and PGR ChIP assays were subjected to standard PCR analyses using specific primers () for specific binding regions. Reverse cross-linked chromatin fragments that were not immunoprecipitated (input DNA) were used as an internal control. […]

Pipeline specifications

Software tools Cistrome, DAVID, GSEA
Databases MSigDB
Organisms Homo sapiens, Caenorhabditis elegans, Mus musculus
Chemicals Progesterone