|
| Status |
Public on Oct 31, 2008 |
| Title |
Poly MCF10AT-YB-1 B |
| Sample type |
RNA |
| |
|
| Source name |
MCF10AT-YB-1 fraction 2
|
| Organism |
Homo sapiens |
| Characteristics |
female; breast cancer cell line MCF10-AT
polysomal fraction mRNA
|
| Treatment protocol |
Total cell extracts from monolayer cultured cells were prepared using the NP-40 lysis buffer containing 20 mM Hepes-KOH, pH 7.6, 100 mM KCl, 5 mM MgCl2, 2 mM dithiothreitol, 0.25% Nonidet P-40, 2 ug/ml leupeptin, 2 ug/ml pepstatin and 10 ug/ml cycloheximide. Cell fractionation, purification of polysomal and post-polysomal mRNA fractions using differential centrifugation on sucrose gradients.
|
| Growth protocol |
Low-passage MCF10AT (also known as MCF10AT1k.cl2) cell lines were provided by Dr. S.J. Kim (National Cancer Institute, Bethesda) and cultured on tissue culture plastic (Falcon) or on Matrigel-coated plates in defined medium, as previously described (Debnath et al., 2003; Janda et al., 2002; Roskelley et al., 1994).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
Biotinlabelled target for the microarray experiment were prepared using 1 ug of total RNA. The RNA was subjected to a rRNA removal procedure with the RiboMinus Human/Mouse Transcriptome Isolation Kit (Invitrogen) and cDNA was synthesized using the GeneChip® WT (Whole Transcript) Sense Target Labeling and Control Reagents kit as described by the manufacturer (Affymetrix).
|
| |
|
| Hybridization protocol |
Following fragmentation, 5 ug of cRNA were hybridized for 16-20 hr at 45C on GeneChip Human Exon 1.0 ST array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
|
| Scan protocol |
GeneChips were scanned using the GeneChip Scanner 3000 7G (Affymetrix).
|
| Description |
Probeset RMA summarized expression data from polysomal fraction mRNA MCF10AT-YB-1 cells, biological replicate B
|
| Data processing |
Genetrix software used for data processing. First the data was normalized using RMA sketch quantile normalization on PSRs with CORE level annotation (~283K PSRs) generating both summarized gene-level and individual normalized PSR-level expression matrices. Gene-level summarized expression data was determined using CORE annotation level PSRs only using the core-mapping file that maps HuExon 1.0 st exon probesets to HG-U133 2.0 probesets (see Series supplementary file).
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| |
|
| Submission date |
Oct 30, 2008 |
| Last update date |
Apr 27, 2009 |
| Contact name |
Elai Davicioni |
| E-mail(s) |
elai.davicioni@veracyte.com
|
| Organization name |
Veracyte, Inc
|
| Street address |
9725 Lusk Blvd
|
| City |
San Diego |
| State/province |
California |
| ZIP/Postal code |
92121 |
| Country |
USA |
| |
|
| Platform ID |
GPL5188 |
| Series (1) |
| GSE13410 |
Mechanism and transcriptional program of YB-1 in breast cancer cell lines. |
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