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Pipeline publication

[…] on of sequencing libraries using Illumina TruSeq Stranded Total RNA/Ribo-Zero Sample Prep Kit. A total of 500–1,000 ng of riboRNA-depleted total RNA was fragmented by RNase III treatment at 37°C for 10–18 min and RNase III was inactivated at 65°C for 10 min. Size selection (50 to 150 bp fragments) was performed using the FlashPAGE denaturing PAGE-fractionator (Thermo Fisher Scientific) prior to ethanol precipitation overnight. The resulting RNA was directionally ligated, reverse-transcribed and RNase H treated., Samples with biological triplicates were sequenced using the Illumina HiSeq2000 platform at the Mayo Clinic Medical Genome Facility. Pre-analysis quality control was performed using FastQC ( and RSeQC software to ensure that raw data are in excellent condition and suitable for downstream analyses. Pair-end raw reads were aligned to the human reference genome (GRch37/hg19) using Tophat. Genome-wide coverage signals were represented in BigWig format to facilitate convenient visualization using the UCSC genome browser. Gene expression was measured using RPKM (Reads Per Kilo-base exon per Million mapped reads) as described previously. EdgeR was used to identify genes that were differentially expressed between EV-expressing and SPOP-F133V-expressing C4-2 cells treated with or without JQ1. Raw and processed data have been deposited into NCBI Gene Expression Omnibus with accession number GSE88872., ChIP was performed as described previously. ChIP-seq libraries were prepared using the methods as described previously and high throughput sequencing was performed using the Illumina HiSeq2000 platforms at the Mayo Clinic Medical Genome Facility. The data were analyzed using the following pipeline: ChIP-seq raw reads were aligned to the human reference genome (GRCh37/hg19) using Bowtie2 (2.2.9), and reads mapped to one or two locations were kept for further analysis, peak calling was performed by MACS2 (2.1.1) with p-value threshold of 1e-5. BigWig files were generated for visualization with the UCSC genome browser or IGV. We used GREAT ( to assign peaks to their potential target genes (a peak-gene association is determined if the peak falls into 2 kb region centering on the transcription start site of the gene). The common BRD4 target genes induced by SPOP F133V and HA-BRD4 expression were determined independently in each of two biological repeat experiments. Raw and processed data have been deposited into NCBI Gene E […]

Pipeline specifications

Software tools FastQC, RSeQC, TopHat, edgeR, Bowtie2
Databases GEO