5 protocols
Sequencing reads were generated by the Lifetech SAETS analysis software. Mapping to the genome reference was performed with the Lifetech Bioscope and Lifescope pipelines and peak calling was performed with the MACS, SICER and SeqSolve programs
A pool of 8 libraries was made using 473 pg of total DNA. In each case, the DNA was added to the PCR mix containing in a final volume of 5.6 mL: PCR Gold Buffer (Applied Biosystems), 1 pi; AmpliTaq Gold, 3000 U; ePCR primer 1, 40 nM; ePCR primer 2, 3 uM; deoxynucleotide mix, 3.5 mM each; MgCl2, 25 mM; 1.6 billion SOLiD sequencing beads (Applied Biosystems). The PCR mix was added to 50 mL SOLiD ePCR Tube containing 9 ml of oil phase and emulsified using Ultraturrax Tube Drive (IKA). The emulsion was dispensed into 96-well plate and amplified for 40 cycles in a 9700 PCR device (Applied Biosystems). After amplification, the emulsion was broken disrupted with butanol, beads were enriched for template positive beads, 3p-end extended and covalently attached onto sequencing slides. A pool of eight samples were deposited on one sequencing slide and sequenced using standard settings on the SOLiD system version 4.0 to produce 50 nucleotide long reads.
nucleic_acid_DNA extraction
Immunoprecipitate DNA from Chromatin Immunoprecipitaion assay (ChIP) using antibody to eNOS (Polyclonal Rabbit Anti-eNOS/NOS Type III from BD Transduction Laboratories, Material Number: 610299).
Immortalized cells from Primary Prostate Carcinoma: ISCOVE medium plus 20% FBS (Hyclone). Cell line from Prostate Carcinoma derived from metastatic site:
Cells were cultured in 150mm dishes for 72 hours in medium supplemented with hormone-deprived serum before addition of 17b-Estradiol 10 -7 M for 45 minutes or veichle (ethanol)