title: 454 GS 20 sequencing; MNase and HpaII sensitivity assays in MCF7 breast cancer cells
design: MNase and HpaII sensitivity assays in MCF7 breast cancer cells
library_construction_protocol: MCF7 cells were cultured in RPMI1640 medium (GIBCO/BRL) supplemented with 10% (v/v) fetal bovine serum (GIBCO/BRL), 2.0 g/L sodium bicarbonate, and were incubated in a humidified 37C incubator with 5% CO2. Cells were washed with ice-cold 1X PBS, and lysed with 700 ul of MNase lysis buffer for 15 min on ice. Cell nuclei were gently rinsed and suspended in 650 ul of MNase digestion buffer (with CaCl2). The reaction was performed by adding 5U of MNase and incubating at 25C for 5 min. Reaction was terminated by adding 40 ul of MNase stop buffer and 20ul of 20% SDS. Suspensions were collected and treated with 0.1 mg/ml of RNase A for an hour at 37C, and then with 0.3 mg/ml of proteinase K for 12-16 hr at 55C. DNA was phenol/chloroform extracted and ethanol-precipitated. The 454 sequencing libraries of MNase treated DNA were constructed using GS 20 DNA Library Preparation Kit (Roche Diagnostics) with the omission of any DNA shearing step and followed the recommended modification for low molecular weight DNA samples. Briefly, DNA fragments ends were polished and ligated with 454 adapters. The adapter-ligated DNA fragments were then immobilized onto streptavidin beads, repaired by a fill-in polymerase followed by alkaline denaturation to isolate single-stranded DNA (ssDNA) library. The quality and quantity of the ssDNA library was assessed using the Agilent 2100 Bioanalyzer.
attributes.tag: Experimental Factor: enzyme
attributes.value: Mnase
attributes.tag: Experimental Factor: fragment size
attributes.value: N/A