title: Illumina HiSeq 2000 paired end sequencing; 5Aza and TSA treatment of MCF7 cells (BS-seq)
design: 5Aza and TSA treatment of MCF7 cells (BS-seq)
library_construction_protocol: MCF7 cells were grown in RPMI 1640 medium upplemented with 10% fetal bovine serum (FBS) and 2.0 g/L sodium bicarbonate at 37°C in humidified incubator (5% CO2). MCF7 cells at logarithmic growth phase were harvested and seeded into 5 cm plate at the density of 5 x 10^6/mL for 24 hours. Two experimental groups were set up: (1) 5Aza (final concentration of 20 uM) for 72 hours. 5Aza-containg medium containing was refreshed every 24 hours. (2) TSA (final concentration of 0.3 uM) for 48 hours. Cells were washed in 1X phosphate buffered saline (PBS) and lysed in cell lysis buffer. Cells were treated with 0.1 mg/mL of RNase A for an hour at 37°C and 0.3 mg/mL Proteinase K for 12 to 16 hours at 55°C. DNA was extracted with phenol/chloroform/isoamyl alcohol (24:25:1) repeatedly until the interface is clean. Equal volume of chloroform was added and the mixture was centrifuged (13,000 g, 10 min). 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 water. The integrity of the extracted DNA was checked by 1.2% (w/v) agarose gel electrophoresis. The concentration of DNA was estimated by ultraviolet spectrophotometry. 3-5 μg of DNA was fragmented by sonication with a Bioruptor (Diagenode) following by adapter ligation using Illumina Pair End DNA Sample Prep kit with the use of methylated adapter (Illumina) according to manufacturer’s instruction. For each sample, three adapter-ligated DNA fragments of 200-300 bp, 300-400 bp, and 400-500 bp were isolated by gel electrophoresis and subjected to bisulfite conversion and PCR enrichment independently to generate three separate libraries for each sample. Bisulfite treatment was performed using an EZ DNA Methylation Kit (Zymo) according to recommended protocols. Three separate PCR were performed for each library using PfuTurbo Cx Hotstart DNA polymerase (Stratagene) and then pooling the enrichment products following by gel purification. The PCR-amplified library was quantified by quantitative PCR and the library size was determined on an Agilent 2100 Bioanalyzer (Agilent) with High Sensitivity DNA chip.
attributes.tag: Experimental Factor: Trichostatin A
attributes.value: compound