Computational protocol: Transcriptomic Profiling and H3K27me3 Distribution Reveal Both Demethylase-Dependent and Independent Regulation of Developmental Gene Transcription in Cell Differentiation

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Protocol publication

[…] For transcriptome expression profiling, the raw reads were trimmed using the FASTX-Toolkit [] and aligned against USCS Homo sapiens hg19 using Tophat (version 2.0.10) []. The aligned reads were assembled with the Cufflinks package, version 2.2.1 []. Differentially expressed genes (DEGs) were identified using Cuffdiff []. All data were normalized according to their fragments per kilobase per million map reads (FPKM) for each gene []. DEGs displaying more than twofold changes in their log2 fold-change were selected for functional annotation using the Database for Annotation, Visualization and Integrated Discovery (DAVID) version 6.7 [, ] and the following parameters: threshold count 5 and enrichment probability (EASE score) 0.1. The raw reads used in this work were deposited into the Gene Expression Omnibus database (GEO, http://www.ncbi.nlm.nih.gov/geo) under accession number SRP045949.For the ChIP-seq analysis, the raw reads were aligned using Bowtie2 version 2.2.1 []. UCSC hg19 was used as the reference index. Significantly enriched regions were identified using the hypergeometric Optimization of Motif EnRichment (HOMER) analysis package []. Default parameters were used for peak calling. The identified peaks were visualized using the UCSC Genome Brower (https://www.genome.ucsc.edu).The JMJD3/UTX inhibition assay and qPCR data were statistically analyzed using SPSS 21.0 (SPSS Inc., Chicago, IL). All experiments were conducted in triplicate, and data are presented as the means ± SE. The data were tested with a one-way ANOVA followed by Tukey’s HSD post hoc test. […]

Pipeline specifications

Software tools FASTX-Toolkit, TopHat, Cufflinks, DAVID, Bowtie2, HOMER
Databases GEO
Application ChIP-seq analysis
Organisms Homo sapiens, Mus musculus
Diseases Carcinoma
Chemicals Tretinoin