Human Jurkat E6-1 lymphocyte T cells (ATCC# TIB-152)
Biomaterial provider
ATCC (American Type Culture Collection)
Treatment protocol
For each experimental replicate (n = 3), 60 ml of cell culture were centrifuged at 1000 x g for 7 min and resuspended in 40 ml of fresh RPMI medium supplemented with FBS. Freshly resuspended cells were inoculated into 35 mm Petri dishes containing 2 ml total volume. Total cell numbers per dish ranged between 4.4 and 10.6 x 106 cells for the biological replicates at each time point. Cells were grown for >12 hr prior to addition of AZA-1 (1 nM final) or equivalent amounts of methanolic vehicle (0.1% final) in order to allow recovery from any stress induced by centrifugation. The cells were harvested for RNA extractions at 24 hr.
Growth protocol
Human Jurkat E6-1 lymphocyte T cells (American Type Culture Collection #TIB-152; Manassas, VA, USA) were grown in RPMI medium supplemented with 10% (v/v) fetal bovine serum (FBS) and maintained in humidified 5%:95% CO2:air at 37 oC. Cells were subcultured every 5 to 7 days with fresh medium by transferring 1 ml of cells to 9 ml of fresh supplemented medium in 75 cm^2 screw cap culture flasks.
Extracted molecule
total RNA
Extraction protocol
Immediately following centrifugation at 1000 x g for 5 min, cells were disrupted by resuspending in 1 ml Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA). All samples were processed according to manufacturer?s protocol. Total RNA was resuspended in DEPC water, purified with a Qiagen RNeasy column (Valencia, CA, USA), and quantified by UV-vis spectroscopy. The RNA was then qualified on an Agilent 2100 Bioanalyzer (Palo Alto, CA, USA) to confirm the yield of high quality RNA.
Label
CY3
Label protocol
Four hundred nanograms of total RNA from each time-matched control and treatment sample were amplified separately and labeled with either Cy3 or Cy5 conjugated CTP (Perkin Elmer, Boston, MA, USA) with a low input linear amplification kit (Agilent Technologies, Palo Alto, CA, USA) according to manufacturer’s protocol. After labeling and clean up, amplified RNA was quantified by UV-vis spectroscopy. One microgram each of Cy3- and Cy5-labeled targets were combined and hybridized to an Agilent whole human genome oligonucleotide microarray (cat.# G4112A) array for 17 hr at 60°C. After hybridization, arrays were washed consecutively in solutions of 6X SSPE with 0.005% N-lauroylsarcosine and 0.06X SSPE with 0.005% N-lauroylsarcosine for 1 minute each at room temperature, followed by a 30 s rinse in Agilent stabilization and drying solution. Biological replicates, including a dye swap, were performed at each time point.
Human Jurkat E6-1 lymphocyte T cells (ATCC# TIB-152)
Biomaterial provider
ATCC (American Type Culture Collection)
Treatment protocol
For each experimental replicate (n = 3), 60 ml of cell culture were centrifuged at 1000 x g for 7 min and resuspended in 40 ml of fresh RPMI medium supplemented with FBS. Freshly resuspended cells were inoculated into 35 mm Petri dishes containing 2 ml total volume. Total cell numbers per dish ranged between 4.4 and 10.6 x 106 cells for the biological replicates at each time point. Cells were grown for >12 hr prior to addition of AZA-1 (10 nM final; 3-fold the 24 hr EC50; Twiner et al. (2005)) or equivalent amounts of methanolic vehicle (0.1% final) in order to allow recovery from any stress induced by centrifugation. The cells were harvested for RNA extractions at 1, 4, and 24 hr.
Growth protocol
Human Jurkat E6-1 lymphocyte T cells (American Type Culture Collection #TIB-152; Manassas, VA, USA) were grown in RPMI medium supplemented with 10% (v/v) fetal bovine serum (FBS) and maintained in humidified 5%:95% CO2:air at 37 oC. Cells were subcultured every 5 to 7 days with fresh medium by transferring 1 ml of cells to 9 ml of fresh supplemented medium in 75 cm^2 screw cap culture flasks.
Extracted molecule
total RNA
Extraction protocol
Immediately following centrifugation at 1000 x g for 5 min, cells were disrupted by resuspending in 1 ml Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA). All samples were processed according to manufacturer?s protocol. Total RNA was resuspended in DEPC water, purified with a Qiagen RNeasy column (Valencia, CA, USA), and quantified by UV-vis spectroscopy. The RNA was then qualified on an Agilent 2100 Bioanalyzer (Palo Alto, CA, USA) to confirm the yield of high quality RNA.
Label
cy5
Label protocol
Four hundred nanograms of total RNA from each time-matched control and treatment sample were amplified separately and labeled with either Cy3 or Cy5 conjugated CTP (Perkin Elmer, Boston, MA, USA) with a low input linear amplification kit (Agilent Technologies, Palo Alto, CA, USA) according to manufacturer’s protocol. After labeling and clean up, amplified RNA was quantified by UV-vis spectroscopy. One microgram each of Cy3- and Cy5-labeled targets were combined and hybridized to an Agilent whole human genome oligonucleotide microarray (cat.# G4112A) array for 17 hr at 60°C. After hybridization, arrays were washed consecutively in solutions of 6X SSPE with 0.005% N-lauroylsarcosine and 0.06X SSPE with 0.005% N-lauroylsarcosine for 1 minute each at room temperature, followed by a 30 s rinse in Agilent stabilization and drying solution. Biological replicates, including a dye swap, were performed at each time point.
Hybridization protocol
Microarrays were imaged using an Agilent microarray scanner. Images were extracted with Agilent Feature Extraction software version A7.5.1 and data analyzed with the Rosetta Resolver 7.0 gene expression analysis system (Rosetta Informatics, Seattle, WA, USA). Using a rank consistency filter, features were subjected to a combination linear and LOWESS normalization algorithm (www.agilent.com). Based on the Rosetta error model designed for the Agilent platform, a composite array was generated at each time point, in which the data for each feature underwent a weighted averaging based on feature quality in the triplicate arrays making up the composite.
Scan protocol
Arrays were scanned with an Agilent microarray scanner at a 10 um scan resolution. Scans were with both the red and green PMT concurrently. Adjustments were made to the PMT intensity as necessary.
