ERS661357 SAMEA3257442 9606 Homo sapiens Protocols: MCF-7 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 37 degrees C incubator with 5% CO2. MCF-7 cells were transfected with H2ac siRNA duplex (a mixture of equimolar concentrations of 5'-CUGCUAGGCCGGGUGACCA-3' and 5'-UGGUCACCCGGCCUAGCAG-3'). Cells transfected with siRNAs were harvested 48 hours post transfection. MCF-7 cells were washed with 1X PBS and resuspended with cell lysis buffer. Cells were treated with 0.1 mg/mL of RNaseA for an hour at 37 degrees C and 0.3 mg/mL proteinase K for 12-16 hr at 55 degrees C. DNA was extracted with an equal volume of phenol/chloroform/isoamyl alcohol mixture (24:25:1). The extraction procedure was repeated until the interface is clean. An equal volume of chloroform was then added and the mixture centrifuged for 10 minutes at 13000x g. Finally, the aqueous phase was removed and precipitated with ethanol. After removal of the supernatant, the DNA pellet was washed with 70% ethanol, air-dried, and dissolved in triple distilled H2O. The integrity of the DNA extracted was checked by 1.2 % (w/v) agarose gel electrophoresis. The concentration of DNA was estimated by ultraviolet spectrophotometry. Bisulfite-seq library was prepared using a method based on Lister et al. Briefly, 3-5 ug of DNA was fragmented by sonication with a Bioruptor (Diagenode, Sparta, NJ) following by adapter ligation using the Pair End DNA Sample Prep kit (Illumina Inc., USA) with the use of methylated adapter (Illumina Inc., USA) according to manufacturer's instruction. For each sample, four adapter-ligated DNA fragments of 200-250 bp, 250-300 bp, 300-350 bp and 350-400 bp were isolated by gel electrophoresis and subjected to bisulfite conversion and PCR enrichment independently to generate 4 separate libraries for each sample. Bisulfite treatment was performed using an EZ DNA Methylation-Gold Kit (Zymo Research) that converts unmethylated cytosines to uracils and leaves methylated cytosines unchanged. Four separate PCR were performed for each library using PfuTurbo(r) Cx Hotstart DNA polymerase (Stratagene) and then pooling the enrichment products following by gel purification. PCR-amplified library was quantified by quantitative PCR and the library size was determined on an Agilent 2100 Bioanalyzer with High Sensitivity DNA chip. ENA-FIRST-PUBLIC 2020-12-31T11:13:54Z ENA-LAST-UPDATE 2018-03-08T23:51:48Z External Id SAMEA3257442 INSDC center name Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan INSDC first public 2020-12-31T11:13:54Z INSDC last update 2018-03-08T23:51:48Z INSDC status public Submitter Id E-MTAB-3320:MCF7-1H2ac-KD broker name ArrayExpress cell line MCF-7 common name human sample name E-MTAB-3320:MCF7-1H2ac-KD scientific_name Homo sapiens