|
| Status |
Public on Aug 19, 2009 |
| Title |
Jurkat Postactivation 12hr HuR-IP rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Jurkat Cells 12hr Postactivation with PMA and PHA
|
| Organism |
Homo sapiens |
| Characteristics |
Jurkat Cells 12hr Postactivation with PMA and PHA, HuR IP
|
| Treatment protocol |
Mitogenic activation: cells were cultured in the presence of 50 ng/ml PMA and 2 μg/ml PHA (Calbiochem/EMD).
|
| Growth protocol |
Human acute T cell leukemia Jurkat cells were cultured in RPMI 1640 supplemented with 10% FBS (GIBCO/BRL).
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
RIPs utilized 100 μL pre-swollen and packed Protein-A Sepharose beads (Sigma) loaded with 30 μg of anti-HuR (3A2), anti-PABP, IgG1 (BD PharMingen) or normal rabbit sera immunoglobulin. Antibody loaded beads were incubated with 3 mg (total protein) cell lysate for four hours at 4ºC, washed 4 times with ice-cold NT2 buffer (50mM Tris pH 7.4/150mM NaCl/1 mM MgCl2/0.05% Nonidet P-40) followed by 3 washes with ice-cold NT2 supplemented with 1M Urea. Extraction of associated RNA was performed as described, and total RNA was isolated using the Trizol reagent (Invitrogen).
|
| Label |
cy3
|
| Label protocol |
RNA from each sample and the reference (Universal Human Reference RNA, Stratagene) were hybridized to oligo (dT) primers at 65º C and then incubated at 42º C for 2 hours in the presence of reverse transcriptase, Cy5- or Cy3-dUTP and Cy5- or Cy3-dCTP, and a deoxynucleotide mix. In all cases, Jurkat derived RNA samples were labeled with Cy3 and reference samples were labeled with Cy5.
|
| |
|
| Channel 2 |
| Source name |
Reference mRNA
|
| Organism |
Homo sapiens |
| Characteristics |
Reference mRNA from Stratagene
|
| Treatment protocol |
Mitogenic activation: cells were cultured in the presence of 50 ng/ml PMA and 2 μg/ml PHA (Calbiochem/EMD).
|
| Growth protocol |
Human acute T cell leukemia Jurkat cells were cultured in RPMI 1640 supplemented with 10% FBS (GIBCO/BRL).
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
RIPs utilized 100 μL pre-swollen and packed Protein-A Sepharose beads (Sigma) loaded with 30 μg of anti-HuR (3A2), anti-PABP, IgG1 (BD PharMingen) or normal rabbit sera immunoglobulin. Antibody loaded beads were incubated with 3 mg (total protein) cell lysate for four hours at 4ºC, washed 4 times with ice-cold NT2 buffer (50mM Tris pH 7.4/150mM NaCl/1 mM MgCl2/0.05% Nonidet P-40) followed by 3 washes with ice-cold NT2 supplemented with 1M Urea. Extraction of associated RNA was performed as described, and total RNA was isolated using the Trizol reagent (Invitrogen).
|
| Label |
cy5
|
| Label protocol |
RNA from each sample and the reference (Universal Human Reference RNA, Stratagene) were hybridized to oligo (dT) primers at 65º C and then incubated at 42º C for 2 hours in the presence of reverse transcriptase, Cy5- or Cy3-dUTP and Cy5- or Cy3-dCTP, and a deoxynucleotide mix. In all cases, Jurkat derived RNA samples were labeled with Cy3 and reference samples were labeled with Cy5.
|
| |
|
| |
| Hybridization protocol |
Sample and reference cDNA were then pooled, purified with QIAquick Purification Columns (Qiagen), mixed with hybridization buffer (50% formamide, 5 SSC, and 0.1% SDS), COT-1 DNA, and poly-deoxyadenylic acid to limit nonspecific binding, and heated to 95º C for 2 minutes. This mixture was pipetted onto a microarray slide, and hybridized overnight at 42º C on the MAUI hybridization system (BioMicro Systems).
|
| Scan protocol |
Arrays were scanned on a GenePix 4000B microarray scanner (Axon Instruments).
|
| Description |
Immunoprecipitated mRNA
Biological Replicate 1 of 3. HuR IP. 12hr Postactivation
|
| Data processing |
All arrays were subject to loess normalization within each array and scale normalization across all arrays using the Array Magic package in R. Replicate probes were collapsed to one probe corresponding to the median value of all the replicates. In order to be considered for subsequent analysis, a probe had to be twofold above local background in all three biological replicates for any of the RIPs or the totals at any time point.
|
| |
|
| Submission date |
Jul 03, 2008 |
| Last update date |
Aug 19, 2009 |
| Contact name |
Neelanjan Mukherjee |
| E-mail(s) |
[email protected]
|
| Phone |
919-684-1632
|
| Organization name |
Duke University
|
| Department |
Institute for Genome Sciences & Policy
|
| Street address |
101 Science Drive
|
| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27708 |
| Country |
USA |
| |
|
| Platform ID |
GPL5770 |
| Series (1) |
| GSE11989 |
Coordinated posttranscriptional mRNA population dynamics during T-cell activation |
|
