7 protocols
AccessionType
bioassay_data_transformation
Data heading descriptions from GEO:
#ID_REF =
#RAW_VALUE = Raw signal from the Feature Extraction software
#VALUE = z-score
hybridization
Cy3 and Cy5 probes were applied to the Human 44K whole genome oligo array kit. Slides were hybridized in a rotating chamber overnight at 60°C, and washed on next day in 6X SSC, 0.005% Triton X-102 for 10 minutes, and then in 0.1X SSC, 0.005% Triton X-102 for 5 minutes on ice. Slides were dried using a nitrogen-filled air gun, and scanned with an Agilent scanner.
labeling
cDNA was made from total mRNA using the BIO-RAD (Hercules, CA) cDNA synthesis kit. Then cRNA was made from cDNA, amplified and labeled with Cy-3 using the Agilent low-input linear amplification kit, according to manufacturer’s protocols.
specified_biomaterial_action
Blood was collected from healthy human donors, PBMCs were isolated from blood using Ficoll gradients and T cells were purified using R&D Systems T cell enrichment columns. T cells were put in six-well plates and treated with 100 ng/ml of CXCL12. Number of cells were always kept equal in terms of chemokines. All of the chambers and plates were incubated for 3 h in a humidified incubator at 37°C with 5% CO2. After incubation, the cells were harvested from the plates for RNA isolation.
nucleic_acid_extraction
Total RNA was isolated by Trizol (Invitrogen, Carlsbad, CA), method using the RNA isolation kit (Quigen, Valencia, CA). RNA quality and quantity were analyzed using the Agilent Bioanlayzer platform (Agilent Technologies, Palo Alto, CA).
specified_biomaterial_action
Blood was collected from healthy human donors, PBMCs were isolated from blood using Ficoll gradients and T cells were purified using R&D Systems T cell enrichment columns. T cells were put in six-well plates and were cultured in media alone. Number of cells were always kept equal in terms of chemokines. All of the chambers and plates were incubated for 3 h in a humidified incubator at 37°C with 5% CO2. After incubation, the cells were harvested from the plates for RNA isolation.
labeling
cDNA was made from total mRNA using the BIO-RAD (Hercules, CA) cDNA synthesis kit. Then cRNA was made from cDNA, amplified and labeled with Cy-5 using the Agilent low-input linear amplification kit, according to manufacturer’s protocols.