|
| Status |
Public on Apr 03, 2015 |
| Title |
B_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Human Brain Reference RNA (HBRR, Ambion) + ERCC Mix2 = Sample B
|
| Organism |
Homo sapiens |
| Characteristics |
seqc sample: B
mixing ratio: 0:1
|
| Extracted molecule |
total RNA |
| Extraction protocol |
mixing of total RNA in known ratios
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 50 ng total RNA using the Agilent kit for One-Color Microarray-Based Exon Analysis Low Input Quick Amp WT labeling (Agilent V 1.0, November 2010) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with a NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
10 µg of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) were fragmented at 70°C for 5 minutes in a reaction volume of 250 µl containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 µl of 2x GExAgilent hybridization buffer were added to the fragmentation mixture and hybridized to Agilent SurePrint Custom GE 1 Million microarrays (Agilent # G4860A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent). The slides were dried after removing them slowly from Wash buffer 2 solution.
|
| Scan protocol |
Microarrays were scanned with an Agilent G2505C Microarray scanner using one color scan settings (scan area 61mmx21.6 mm) at a resolution of 2 µm in double pass scanning. The dye channel was set to Green and Green PMT was set to 100% to yield a high-sensitivity 20 bit TIFF image. A second scan at a lower PMT yield proved unnecessary for these hybridizations.
|
| Description |
MAQC-III sample B
|
| Data processing |
Agilent Feature Extraction Software (v10.10.1.1) was applied with the default parameters appropriate for the chip type (1M feature array; protocol GE1_1010_Sep10 and Grid 039825_D_F_20120329), unprocessed median spot intensities without background subtraction were exported. We applied the published HOOK algorithm for saturation detrending and the FARMS algorithm for expression level estimates from probe groups.
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| |
|
| Submission date |
Jun 17, 2013 |
| Last update date |
Apr 03, 2015 |
| Contact name |
Pawel P. Labaj |
| Organization name |
Boku University Vienna
|
| Department |
Department of Biotechnology
|
| Lab |
Chair of Bioinformatics
|
| Street address |
Muthgasse 18
|
| City |
Vienna |
| ZIP/Postal code |
1190 |
| Country |
Austria |
| |
|
| Platform ID |
GPL17298 |
| Series (2) |
| GSE47792 |
SEQC Project |
| GSE48016 |
MAQC-III/SEQC Main Study ABCD titration experiment, ERCC spike-in profiles and gene-level estimates for a selected test set |
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