title: Illumina HiSeq 2000 paired end 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 three times with ice-cold 1X PBS on culture dish. The cells were lysed with 700 ul of ice-cold TZN buffer (10 mM Tris-HCl pH 7.6, 0.2 mM ZnCl2, 0.2% NP-40) on ice for 15 min. The lysate was removed by suction, and then gently rinsed the nuclei with 700ul of 1X NEB buffer 1. The reaction was performed by incubating cell nuclei in 650 ul of 1X NEB buffer 1 and 50U of HpaII at 37C for 6hr. To fill in and mark the DNA ends, 30 uM dATP, dGTP, dTTP, biotin-14-dCTP (Invitrogen), and 10 ul 5U/ul Klenow (NEB) were added to cell nuclei. The mixtures were incubated at 37C for 1 hr and subsequently placed on ice. The reaction was terminated by added 40 ul stop buffer (100 mM EDTA and 10 mM EGTA) and 20 ul of 20% SDS. Suspensions were collected in a 1.5 ml Eppendorf tube and treated with 0.3 mg/ml of proteinase K for 12-16 hr at 65C. Samples were extracted with equal volumes of phenol/chloroform/isoamyl alcohol mixture (24:25:1), the extraction procedure was repeated until the interface was clean. An equal volume of chloroform was then added, and the solution was centrifuged for 10 min at 13,000g. The aqueous phase was ethanol-precipitated, and the DNA pellet was washed with 70% ethanol, air-dried, and dissolved in d3H2O. 2-3 ug of HpaII treated and biotin labeled DNA was fragmented by sonication with a Bioruptor (Diagenode, Sparta, NJ) and size-fractionated by 2% agarose gel. Two biotinylated DNA fragments of 300-500 bp and 500-800 bp were purified by Dynal magnetic M-280 streptavidin beads and subjected to library construction independently by performing end-repair reaction, addition 3'-A to the ends and adapter ligation on the biotinylated DNA immobilized to the streptavidin beads with the use of TruSeq DNA Sample Preparation Kit (Illumina Inc., USA). Two separate PCR of 18 cycles were performed for each fragment libraries using KAPA HiFi DNA Polymerase (Kapa Biosystems) and then pooling the enrichment products followed by purifying with AMPure XP Beads. PCR-amplified libraries were quantified by quantitative PCR and the library size was determined on an Agilent 2100 Bioanalyzer.
attributes.tag: Experimental Factor: enzyme
attributes.value: HpaII
attributes.tag: Experimental Factor: fragment size
attributes.value: 500-800 bp