Illumina HiSeq 2000 sequencing; 5Aza and TSA treatment of MCF7 cells (RNA-seq)
5Aza and TSA treatment of MCF7 cells (RNA-seq)
MCF7 cells were grown in RPMI 1640 medium upplemented with 10% fetal bovine serum (FBS) and 2.0 g/L sodium bicarbonate at 37Â°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. 'MCF7 cells were grown to 85% confluence in 6 cm tissue culture dish. Cells were washed twice with 1X PBS. Total RNA was extracted using TRI Reagent (Invitrogen) according to the manufacturer''s protocol. The RNA quality was assessed by 1.2% (w/v) agarose gel electrophoresis and quantified by a UV spectrophotometer.' 5 micro-g of total RNA with RIN (RNA integrity number) ranged from 8.0 to 10 was subjected to the removal of rRNA using Ribo-Zero Gold kit for human/mouse/rat (Epicentre). The sequencing library for rRNA-depleted RNA was then prepared using TruSeq Stranded mRNA Sample Prep Kit (Illumina) which produced directional RNA-seq libraries. Briefly, the rRNA-depleted RNA was fragmented and first-strand cDNA was synthesized using random hexamers with SuperScript III (Invitrogen). The second-strand cDNA was synthesized with dTTP replaced by dUTP to generate double-stranded cDNA following by end repair, addition of a single A base and adapter ligation. The adapter-ligated double-stranded cDNA library was enriched with 6 to 10 cycles of PCR using KAPA HiFi DNA polymerase (Kapa Biosystems). The RNA libraries were quantified by qPCR and quality tested using Agilent 2100 Bioanalyzer with High Sensitivity DNA chip.
Experimental Factor: Trichostatin A
compound