|
Status |
Public on Jul 29, 2008 |
Title |
pLXSN/HuR/3 |
Sample type |
RNA |
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Source name |
Breast epithelial cells MCF10A, RNA from IP reactions using an anti-HuR antibody and pLXSN empty vector transfected cells.
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Organism |
Homo sapiens |
Characteristics |
RNA from IP reactions using an anti-HuR antibody and pLXSN empty vector transfected cells. Biological repeat 3.
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Treatment protocol |
pLXSN empty vector stable transfected cells
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Extracted molecule |
total RNA |
Extraction protocol |
After IP with HuR antibody, beads were incubated with 100 ul NT2 buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.05% NP-40) containing 20 U RNase-free DNase I for 5 min at 37°C, washed with NT2 buffer, and incubated in 100 ul NT2 buffer containing 0.1% SDS and 0.5 mg/ml proteinase K (15 min, 55°C). RNA was extracted using acid phenol-ChCl (Ambion) and precipitated in the presence of glycoblue.
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Label |
biotin
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Label protocol |
standard Illumina protocol using Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791) In short, 0.5g of total RNA was first converted into single-stranded cDNA with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules. The double stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in vitro transcription reaction where single-stranded RNA (cRNA) was generated and labeled by incorporation of biotin-16-UTP.
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Hybridization protocol |
standard Illumina protocol. In short, a total of 0.75 ug of biotin-labeled cRNA was hybridized at 58 degrees C for 16 hours to Illumina's Sentrix HumanRef-8 Expression BeadChips (Illumina, San Diego, CA). Each BeadChip has 24,000 well-annotated RefSeq transcripts with approximately 30-fold redundancy. The arrays were washed, blocked and the labeled cRNA was detected by staining with streptavidin-Cy3.
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Scan protocol |
Arrays were scanned at a resolution of 0.8um using the Beadstation 500 X from Illumina.
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Description |
Breast epithelial cells MCF10A
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Data processing |
Data was extracted using the Illumina BeadStudio software(v1). Any spots at or below the background were filtered out using an Illumina detection score of 0.98 and below. The natural log of all remaining genes were used to find the avg and std of each array and the z-score normalization was calculated and presented below. Z-score = (raw value - avg)/std. Complete data including detection scores included in the supplementary file..
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Submission date |
Jul 23, 2008 |
Last update date |
Jun 22, 2020 |
Contact name |
Supriyo De |
Organization name |
NIA-IRP, NIH
|
Department |
Laboratory of Genetics and Genomics
|
Lab |
Computational Biology & Genomics Core
|
Street address |
251 Bayview Blvd
|
City |
Baltimore |
State/province |
Maryland |
ZIP/Postal code |
21224 |
Country |
USA |
|
|
Platform ID |
GPL6102 |
Series (1) |
GSE12215 |
Identification of transformation-related pathways in a breast epithelial cell model using a ribonomics approach. |
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