title: NextSeq 500 paired end sequencing; RNA seq of prostate gland from WT, PTEN pc -/- and PTEN pc-/-;P53 -/- mice
design: RNA seq of prostate gland from WT, PTEN pc -/- and PTEN pc-/-;P53 -/- mice
library_construction_protocol: For wild-type, Ptenpc-/- and Ptenpc-/-; Trp53pc-/-, three mice of 20 weeks of age were analyzed. Prostate tumor tissues were quickly excised and homogeneized in polysome lysis buffer (30 mM Tris-HCl, pH 7.5, 100 mM NaCl, 30 mM MgCl2, 1% sodium deoxycolate, 1% Triton X-100, 100 mg ml−1 cycloheximide, 1 mM DTT and 30 U ml−1 RNasin) by using a sterile pestle. RNA was extracted by phenol/chloroform 5:1  (Sigma-Aldrich) method, followed by overnight isopropanol precipitation at -80 C, centrifugation at 14000 r.p.m. for 45 minutes and resuspension in 10 ul of DEPC water. At the end of the procedure, DNAse digestion was carried out with Turbo DNase (Thermo Scientific) for 30 minutes at 37 C and RNA purification was performed with RNA Clean and Concentrator kit (Zymo research) Whole library preparation was performed starting from 1oo ng of RNA.