DNA content and cell cycle were selleck Gemcitabine then analyzed using the software program MultiCycle (Phoenix Flow Systems, San Diego, CA). DNA Extraction DNA from sorted nuclei was extracted using an amended protocol from QIAamp? DNA Micro Kit from Qiagen (Valencia, CA). Briefly each sorted sample was resuspended in 180 ��l buffer ATL and 20 ��l proteinase K then incubated for 3 hours at 56��C for complete lysis. Samples were bound and washed according to QIAamp? DNA Micro Kit instructions, eluted into 50 ��l of H20, then precipitated overnight with 5 ��l 3 M sodium acetate and 180 ��l 100% EtOH. Each sample was then centrifuged for 30 minutes at 20,000��g, washed in 1 ml of 70% EtOH for 30 minutes at 20,000��g. The samples were carefully decanted and the DNA pellet was dried by speed vacuum then resuspended in a small volume (e.
g. 10�C50 ��l) of H2O for final concentrations suitable for accurate quantification. DNA Amplification Genomic DNAs from sorted FFPE samples were amplified using Ovation? WGA FFPE System from NuGEN? Technologies (San Carlos, CA). DNA was processed in accordance with Ovation? WGA FFPE standard SPIA protocol with an alternate T7 endonuclease fragmentation step. Resulting amplified product was either used as template for aCGH analysis or processed with the Nugen Encore ds-DNA module according to the supplier��s instructions in order to generate double-stranded (ds) end repaired DNA as input for library suitable for next generation sequencing. Extracted fresh frozen sourced genomic DNA was amplified using the phi29 based Illustra GenomiPhi V2 Amplification kit from GE Healthcare Bio-sciences Corp (Piscataway,NJ) according to our published protocols [14].
A 100 ng aliquot of pooled 46, XX DNA (Promega, Madison, WI) was amplified with the matching amplification protocol to generate a suitable reference for each aCGH experiment using amplified DNA template. In all cases the quality of the amplification product was assessed by gel electrophoresis. aCGH Analysis Fresh frozen phi29 amplified and FFPE non-amplified DNAs were treated with DNAse 1 prior to Klenow based labeling. High molecular weight phi29 templates were digested for 30 minutes while the smaller fragmented FFPE samples were digested for only 1 minute. In each case 1 ��l of 10�� DNase 1 reaction buffer and 2 ��l of DNase 1 dilution buffer were added to 7 ��l of DNA sample and incubated at room temperature then transferred to 70��C for 30 minutes to deactivate DNase 1. In contrast the amplified Brefeldin_A FFPE sourced DNAs do not require DNase 1 treatment prior to Klenow-based labeling. Sample and reference templates were then labeled with Cy-5 dUTP and Cy-3 dUTP respectively using a BioPrime labeling kit (Invitrogen, Carlsbad, CA) according to our published protocols [14].