Computational protocol: Intrinsic BET inhibitor resistance in prostate cancer caused by SPOP mutation-mediated BET protein stabilization

Similar protocols

Protocol publication

[…] For whole genome sequencing, DNA was extracted by phenol-chloroform and purified by the ethanol precipitation method from 32 paired tumor and benign frozen patient samples. DNA samples were fragmented in fragmentation buffer using Covaris Ultrasonicator system. The fragmented DNA with average length of 500 bp was subjected to DNA library construction. Libraries were constructed according to Illumina’s protocol with DNA samples. High-throughput short-gun sequencing was performed on the IlluminaHiSeq 2000 platform. For DNA sequencing, pair-end reads with length of 90 bp were generated. Raw reads of DNA sequencing were filtered using an in-house pipeline. Clean DNA reads were processed with SAMTools to remove the PCR duplicates and aligned to the human reference genome hg19 with Burrows-Wheeler Aligner (http://bio-bwa.sourceforge.net/). The whole genome sequencing data have been deposited in The European Genome-phenome Archive with the accession # EGAS00001000888.For Sanger sequencing, DNA was extracted from all 99 cases of FFPE prostate cancer tissues using a QIAamp DNA FFPE Tissue kit. PCR was performed and PCR products were purified using a GeneJET Extraction kit according to manufacturer’s instruction and used for Sanger sequencing. The primers used for DNA amplification were: Amp-Exon6-Forward 5′-ACCCATAGCTTTGGTTTCTTCTCCC-3′; Amp-Exon6-Reverse 5′-TATCTGTTT TGGACAGGTGTTTGCG-3′; Amp-Exon7-Forward 5′-ACTCATCAGATCTGGGAACTGC-3′; Amp-Exon7-Reverse 5′-AGTTGTGGCTTTGATCTGGTT-3′. Amp-Exon6-Reverse and Amp-Exon7-Forward were also used for Sanger sequencing. [...] C4-2 cells infected with lentivirus expressing empty vector (EV), HA-SPOP-F133V or BRD2/3/4 were treated with or without JQ1 (1 μM) for 24 h. Total RNAs were isolated from cells using the methods as described previously. Briefly, RNA was isolated using RNeasy Plus Mini Kit (Qiagen). High quality (Agilent Bioanalyzer RIN >7.0) total RNAs were employed for the preparation 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 (http://www.bioinformatics.babraham.ac.uk/projects/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. […]

Pipeline specifications

Software tools FastQC, RSeQC, TopHat, edgeR
Databases GEO UCSC Genome Browser
Application RNA-seq analysis
Organisms Homo sapiens
Diseases Prostatic Neoplasms
Chemicals Cholesterol