Valproic acid (VPA) (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in water at concentration of 50mg/ml. After an initial 24 hours of pre-incubation, cells were exposed to VPA at a concentration of 400µg/ml and the culture was continued for an additional 2 days.
Growth protocol
Human CD34+ cells were purified upon donor’s informed consent from umbilical Cord Blood (CB) samples, collected after normal deliveries, according to the institutional guidelines for discarded material. Mononuclear cells were isolated by Ficoll-Hypaque (Lymphoprep; Nycomed Pharma, Oslo, Norway) gradient separation, washed twice with PBS, and then CD34+ cells separated using magnetic cell sorting procedure (EasySep Human CD34+ positive selection kit, StemCell Techonologies Inc.; Vancouver, Canada). CD34+ cell purity assessed by flow cytometry was always >95%. CD34+ hematopoietic cells were seeded in 24-well plates at a density of 5x105cell/ml in Iscove’s modified Dulbecco’s medium (IMDM) (Euroclone, Celbio, Milano, Italy) supplemented with 20% heat inactivated human serum (Lonza, Milano, Italy), 1mM L-Glutamine (Euroclone), SCF (50ng/ml), Flt3-ligand (Flt3-l) (50ng/ml), TPO (20ng/ml), IL-6 (10ng/ml) and IL-3 (10ng/ml) (all from R&D Systems, Minneapolis, MN, USA). CD34+ cells were initially maintained in the media described above and incubated at 37ºC in humidified atmosphere containing 5% CO2.
Extracted molecule
total RNA
Extraction protocol
Total cellular RNA was extracted 48 hours of treatment from 0.5x106 cells of each sample using RNeasy Micro kit (Qiagen, Valencia, CA) following the protocol supplied by the manufacturer. Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit, Agilent Technologies, Waldbrunn, Germany) were used to determine the purity/integrity of RNA samples using Agilent 2100 Bioanalyzer. NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) was used to evaluate the RNA sample concentration, and 260/280 and 260/230 nm ratios were considered to verify the purity of total RNA. RNAs originating from five different experiments were pooled in order to obtain at least 2 µg per sample.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard One Cycle Affymetrix protocol from 2 ug total RNA (Gene Chip Expression Analysis Technical Manual, 701024 Rev3, Affymetrix). Labeled cRNA was purified using Affymetrix GeneChip Sample Cleanup Module and fragmented (15 µg) as described in the Affymetrix GeneChip protocol. Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit, Agilent Technologies, Waldbrunn, Germany) and Agilent 2100 Bioanalyzer were used to determine the cRNA concentration/quality as well as to optimize the cRNA fragmentation.
Hybridization protocol
Following fragmentation, 15 ug of cRNA were hybridized for 16 hr at 45C on Affymetrix HG-U133A GeneChip arrays. GeneChips were washed and stained in the Affymetrix Fluidics Station 450 using the instrument’s standard FS450_0004 protocol with antibody-mediated signal amplification and GeneChip Hybridization, Wash and Staining Kit.
Scan protocol
GeneChip were finally scanned using the Affymetrix GeneChip scanner 3000, enabled for High-Resolution Scanning.
Description
Gene expression data from VPA-treated human CD34+ HSCs from umbilical Cord Blood (CB)
Data processing
The GeneChip-Operating-Software (GCOS) absolute analysis algorithm was used to determine the mRNA expression levels (Signal), while the GCOS comparison analysis algorithm was used in order to compare gene expression levels between two samples. The data were analyzed with GCOS using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 100.
