Hiseq 2500 genome analyzer

Omni ATAC-seq was performed following protocol as previously described 47 (link). About 50,000 viable LNCaP cells (growing in 5% CSS) after GSK2879552 treatment were centrifuged at 500 RCF at 4°C. The pellet was lysed in 50 μl cold resuspension buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl₂ 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin). The lysis solution was then diluted with 1 ml cold buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.1% Tween-20). Nuclei were collected by centrifuged at 500 RCF at 4°C for 10 minutes. Pellet was resuspended in 50 μl of transposition mixture (25 μl 2× TD buffer, 2.5 μl transposase, 16.5 μl PBS, 0.5 μl 1% digitonin, 0.5 μl 10% Tween-20, 5 μl H₂O) using Illumina Tagment DNA TDE1 Enzyme and Buffer Kit, and incubated at 37°C for 30 minutes in a thermomixer with 1,000 RPM mixing. DNA samples were cleaned immediately by Qiagen QIAquick Purification Kit and PCR Pre-amplified by NEBNext High-Fidelity 2× PCR Master Mix. qPCR amplification was used to determine the additional cycles to prevent over-amplification. The final PCR product was purified by Qiagen QIAquick Purification Kit and run on Agilent High Sensitivity Screen Tape for quality control. The libraries were sequenced on the HiSeq 2500 Illumina Genome Analyzer.

RNA was extracted with TRIzol Reagent (Invitrogen) based on the manufacturer’s protocol. For tissue RNA isolation, the RNeasy Mini kit (Qiagen) was used following the manufacturer’s protocol. Same amount of tissue samples were bead (5 mm) milled using TissueLyser LT (Qiagen). Gene expression was measured using realtime RT-PCR analyses with TaqMan one-step RT-PCR reagents on the QuantStudio 3 Real-time PCR system and results were normalized to co-amplified GAPDH. For RNA-seq analysis, RNA was purified using RNeasy Mini kit (Qiagen). TruSeq® Strnd Total RNA LT (Illumina) was used for library construction based on manufacturer’s protocol, and sequencing was performed on HiSeq 2500 Illumina Genome Analyzer.