LNCaP + TNF 48hr Targeted Genome Capture rep1
Genomic DNA was isolated from the cell lines after various treatments using the AllPrep DNA/RNA Mini Kit (Qiagen, #80204) according to the manufacturer's instructions. Genomic DNA libraries were generated following the Illumina Paired-End Genomic DNA Sample preparation protocol. Briefly, 3 micrograms of each genomic DNA was sheared using a Covaris S2 (Covaris Inc, Woburn, MA) to a peak target size of 350 base pairs. The sheared DNA was concentrated using AMPure XP beads (Beckman Coulter), and their ends were repaired using T4 DNA polymerase, Klenow polymerase, and T4 polynucleotide kinase. After 3' A-tailing with exo-minus Klenow polymerase, Illumina paired end adapters were ligated to the genomic DNA fragments. The adapter-ligated libraries were size selected using three percent Nusieve 3:1 (Lonza) agarose gels, where fragments (~ 500bps) were excised and purified with QIAEX II gel extraction reagents (Qiagen). Subsequently the DNA was amplified for 9 cycles using Illumina primers and in that process, barcoded. Following the final round purification with AMPure XP beads, the DNA concentration was determined using a Nanodrop spectrophotometer.Towards target enrichment, we utilized 1ug of the genomic DNA library to setup capture with custom designed SeqCap_EZ library (Roche Nimblegen Inc, Madison, WI) at 47 degrees C for 72 hours using the SeqCap EZ hybridization and wash kit (Roche Nimblegen Inc, Madison, WI) following manufacturer's instruction. The probes designed for this study captured TMPRSS2 (chr21:42,826,001-42,910,000), ERG (chr21:39,732,612-40,072,611) and some control loci (e.g. probes spanning the MIPOL1 and DGKB genes as an internal control for LNCaP cells that harbor a rearrangement between these two genes) (Maher et al., 2009; Tomlins et al., 2007). The targeted genomic fragments were captured by incubation with Dynal M-280 streptavidin beads (Invitrogen).  The beads were then washed strictly following the protocol described in the SeqCap EZ hybridization and wash kit (Roche Nimblegen Inc, Madison, WI), and utilized an additional 8 cycle amplification with the corresponding Illumina primers. The libraries were purified with AMPure XP beads and their quality and quantity were analyzed using an Agilent Bioanalyzer. The paired-end libraries were sequenced at 2 _ 100-nucleotide read length with the Illumina HiSeq 2000 system. Only reads that passed the chastity filter of Illumina BaseCall software were used in subsequent analysis.