Computational protocol: KDM5 lysine demethylases are involved in maintenance of 3′UTR length

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Protocol publication

[…] The sequencing reads (50 bp, single-end) were trimmed for 3′ adapter sequences using Trimmomatic, and the reads shorter than 36 bp after trimming were excluded (). The remaining sequencing reads were aligned to the S. cerevisiae reference genome (SacCer3) using bowtie (). Up to one mismatch was allowed in the 28-bp seeding region for each aligned read. Reads mapping to multiple sites were excluded. ChIP-seq data were deposited in the NCBI Gene Expression Omnibus database under accession number GSE67212. Binding regions were identified using SICER with the following parameters: a effective genome size of 0.74, a window size of 200 bp, and a gap size of 200 bp (). HOMER was used for peak annotation and coverage calculation (). The IGV was used for visualization (). Python and R scripts were used to obtain the metagene plots on the basis of the peak annotation and normalized coverage. The base pair coverage for each gene in the gene set was aggregated by sum at each position of the metagene region (±1000 bp relative to TSSs or polyA sites). The coverage was then normalized to the total coverage in the region for each sample (IP, input separately), followed by a log2 ratio calculation between IP and input. [...] A biotinylated RNA 20-mer was designed using a random sequence generator and obtained from Integrated DNA Technologies. Three RNA structure prediction software tools (RNAfold, Sfold, and RNA structure) were used to determine the absence of secondary structure. Reactions were prepared containing 0.01 μg of RNA and 5.0 μg of recombinant His-Jhd2 in binding buffer [50 mM tris-HCl (pH 7.9), 10% glycerol, 100 mM KCl, 5 mM MgCl2, 10 mM β-mercaptoethanol, and 0.1% NP- 40]. A control that contained no RNA was prepared. The reactions were incubated with rotating for 1 hour at room temperature. Streptavidin-coupled magnetic M-280 Dynabeads (Invitrogen) were added to the reaction, and samples were incubated while rotating for an additional 30 min at room temperature. The beads were washed four times with binding buffer. Samples were combined with SDS loading buffer and incubated at 95°C for 5 min. The proteins were resolved using SDS-PAGE and detected via Western blot using a Jhd2 antibody (Active Motif, 39263). […]

Pipeline specifications

Software tools RNAfold, Sfold
Application RNA structure analysis
Organisms Saccharomyces cerevisiae, Homo sapiens
Diseases Breast Neoplasms