Preparation of biotin-labeled cRNA; RNA samples were quality controlled using the Agilent 2100 Bioanalyzer. 100 pmol T7-(dT)24 primer (5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24-3') was hybridized with the Total RNA ( 8 - 12ug) at 70°C for 10 minutes. First strand cDNA was synthesized by reverse transcription using 400 units SuperScript II Reverse Transcriptase in a reaction containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol (DTT) and 0.5 mM dNTPs at 42C for 1 hour. The reaction was then put on ice for 2 mins, followed by second strand cDNA synthesis. 130ul of the second strand reaction mix was added to the 20ul first strand synthesis reaction. The second strand reaction mix contained 40 units E. coli DNA polymerase I, 10 units E. coli DNA ligase, 2 units E. coli RNase H and 0.2 mM dNTPs in a buffer containing 20 mMTris-HCl (pH 6.9), 90 mM KCl, 4.6 mM MgCl2, 10mM (NH4)SO4 and 0.15 mM ß-NAD+. The reaction was incubated at 16 C for 2 hours. 10 units T4 DNA Polymerase was added and the reaction incubated for a further 5 minutes at 16 C, then the reaction was terminated using EDTA. The double-stranded cDNA was cleaned up using the cDNA Cleanup Spin Column supplied in the Affymetrix GeneChip Sample Cleanup Module. The double-stranded cDNA was used as a template for in Vitro transcription of biotin-labeled cRNA using the ENZO BioArray RNA Transcript Labeling Kit supplied by Affymetrix. The Transcript Labeling kit produces large amounts of hybridizable biotin-labeled RNA targets by in Vitro transcription from bacteriophage T7 RNA polymerase promoters. The reaction mixture containing Biotin-Labeled ribonucleotides (ATP, GTP, CTP, UTP with Bio-UTP and Bio-CTP), DTT, RNase inhibitor mix and T7 RNA polymerase was incubated at 37 C for 5 hrs, gently mixing every 30 minutes during the incubation. This produced an average of 40ug biotin-labeled cRNA . The biotin-labeled cRNA was cleaned up using the cRNA Cleanup Spin Column supplied in the Affymetrix GeneChip Sample Cleanup Module. The quantity and quality of the cRNA was measured using the Agilent 2100 Bioanalyzer. The total amount of cRNA was calculated, the starting quantity of RNA subtracted to give the quantity of adjusted cRNA. The cRNA was fragmented by metal-induced hydrolysis to break down full-length cRNA to 35-200 base fragments. 15ug of adjusted cRNA was used to prepare 300ul of hybridization cocktail of which 200ul was hybridized with the GeneChip. ; Protocol Type = labeling; Performers: John,Y,Okyere, Janet,,Higgins
Hybridization protocol
EukGE-WS2v4; Protocol Type = Hybridization; Parameter Wash A1 Recovery Mixes = 0 (type integer); Parameter Wash A1 Temperature (C) = 25 (type integer); Parameter Number of Wash A1 Cycles = 10 (type integer); Parameter Mixes per Wash A1 Cycle = 2 (type integer); Parameter Wash B Recovery Mixes = 0 (type integer); Parameter Wash B Temperature (C) = 50 (type integer); Parameter Number of Wash B Cycles = 4 (type integer); Parameter Mixes per Wash B Cycle = 15 (type integer); Parameter Stain Temperature (C) = 25 (type integer); Parameter First Stain Time (seconds) = 600 (type integer); Parameter Wash A2 Recovery Mixes = 0 (type integer); Parameter Wash A2 Temperature (C) = 25 (type integer); Parameter Number of Wash A2 Cycles = 10 (type integer); Parameter Mixes per Wash A2 Cycle = 4 (type integer); Parameter Second Stain Time (seconds) = 600 (type integer); Parameter Third Stain Time (seconds) = 600 (type integer); Parameter Wash A3 Recovery Mixes = 0 (type integer); Parameter Wash A3 Temperature (C) = 30 (type integer); Parameter Number of Wash A3 Cycles = 15 (type integer); Parameter Mixes per Wash A3 Cycle = 4 (type integer); Parameter Holding Temperature (C) = 25 (type integer); Software: type: Acquisition and analysis software; Hardware: type: Fluidics Station, Parameter: Parameter Station Number = 1 (type integer), Parameter: Parameter Position = 4 (type integer); Performers: John,Y,Okyere, , Janet,,Higgins
Scan protocol
Protocol Type = CEL Analysis; Parameter Algorithm name = Percentile (type string); Parameter Upper left corner x coordinate = 232 (type integer); Parameter Upper left corner y coordinate = 230 (type integer); Parameter Upper right corner x coordinate = 4502 (type integer); Parameter Upper right corner y coordinate = 205 (type integer); Parameter Lower right corner x coordinate = 4529 (type integer); Parameter Lower right corner y coordinate = 4475 (type integer); Parameter Lower left corner x coordinate = 259 (type integer); Parameter Lower left corner y coordinate = 4500 (type integer); Parameter Percentile = 75 (type integer); Parameter Cell margin = 2 (type integer); Parameter Outlier high = 1.500 (type float); Parameter Outlier low = 1.004 (type float); Software: type: Acquisition and analysis software; Protocol Type = Scan; Parameter Pixel Size = 3 (type integer); Parameter Number of Scans = 2 (type integer); Software: type: Acquisition and analysis software; Hardware: type: Scanner, Parameter: Parameter Scanner ID = not specified (type string), Parameter: Parameter Scanner Type = HP (type string); Performers: John,Y,Okyere, , Janet,,Higgins
Description
Rows (integer) = 712 Cols (integer) = 712 Number of Probe Sets (integer) = 22810 Background Avg (float) = 103.98 Background Stdev (float) = 1.95 Background Max (float) = 110.8 Background Min (float) = 100.0 Noise Avg (float) = 5.74 Noise Stdev (float) = 0.34 Noise Max (float) = 6.8 Noise Min (float) = 5.2 RawQ (float) = 3.81 Number Cells Masked (integer) = 0 Number Outlier Cells (integer) = 462 Number Cells Modified (integer) = 0 Rows (integer) = 712 Cols (integer) = 712 Number of Cells (integer) = 506944 Image: MC002_ATH1_A8.2-dubos-aih.DAT
