Computational protocol: Genome-wide analysis of YB-1-RNA interactions reveals a novel role of YB-1 in miRNA processing in glioblastoma multiforme

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Protocol publication

[…] The iCLIP assay was carried out as described (). Briefly, one 10 cm dish of U251-MG cells were irradiated with UV light at 150 mJ/cm2. After cell lysis, RNAs were partially fragmented using 10 μl high (1:50) or low (1:5000) dilutions of 5 μg/μl RNase A (QIAGEN) followed by immunoprecipitation with 10 μg anti-YB-1 antibody (Sigma) immobilized on protein A Dynabeads (Life Technologies). RNAs were then ligated at 3′ ends to a DNA adapter and radioactively labeled by T4 polynucleotide kinase (Fermentas), and the protein–RNA complex was transferred to a nitrocellulose membrane. For iCLIP cDNA library preparation, RNAs were purified and reverse-transcribed with a primer containing barcode. The resulting cDNAs were purified by PAGE, circularized by single-stranded DNA ligase (Epicentre), linearized by restriction enzyme cleavage, and amplified by PCR.The iCLIP cDNA library was processed on an Illumina HiSeq 2000 sequencer. Mapping of sequence reads was performed against the human genome (version Hg19/NCBI37) allowing one mismatch and single hits using Bowtie (). Identification of clustered crosslink sites was performed as described (,). To analyze the enriched motif, the 21 nt-long sequences were extracted by extending 10 nt from the crosslink sites in both directions. The z-score for each hexamer in the 21 nt region was calculated relative to control sequences randomly selected from similar background. The top 20 motifs with the highest z-scores were performed the multiple sequence alignment using CLUSTALW. The output file was submitted to the WebLogo3 to generate the sequence logo. To investigate whether YB-1 binding sites are enriched on any pre-miRNAs, the iCLIP cDNA counts were normalized to the expression levels of individual mature miRNA detected by small RNA-seq analysis. [...] The iCLIP cDNA counts for YB-1 crosslink sites could be viewed using IGV tool ( by uploading a tdf file from [...] The short reads of small RNA-seq data were processed by cutadapt () to trim the adaptor sequences, and aligned to human genome (hg19) by bowtie () using the parameter of ‘-v1 –best –strata –k11 –m10’. We used the BEDTools () and the GFF3 file downloaded from miRBase () to quantify the aligned reads on each miRNA. The expression values of miRNAs were normalized by the total number of aligned reads in each library. […]

Pipeline specifications

Software tools Bowtie, Clustal W, WebLogo, IGV
Applications CLIP-seq analysis, Genome data visualization
Organisms Homo sapiens
Diseases Glioblastoma, Neoplasms
Chemicals Nucleotides