|
| Status |
Public on Jul 22, 2014 |
| Title |
LL34 targeting shRNA, biological rep2 |
| Sample type |
RNA |
| |
|
| Source name |
MLE12 cells
|
| Organism |
Mus musculus |
| Characteristics |
treatment: LL34 shRNA
|
| Treatment protocol |
Lentivirus was produced in HEK-293T cells in 100-mm cell culture dishes were co-transfected with 3 µg psPAX2, 1 µg pMD2.G, and 4 µg pLKO.1 bearing specific shRNAs. Cells were incubated in transfection medium (DMEM; Gibco, Grand Island, NY) for 12 h, followed by incubation in DMEM supplemented with 10% fetal bovine serum, for 36 h. The culture medium containing lentivirus particles was collected, purified using Amicon Ultra-4 100k centrifugal filters (Millipore, Billerica, MA), and reconstituted in an equivalent volume of MLE12 media. MLE12 cells at 60% confluency were infected with lentivirus-bearing specific shRNAs in a 1:4 ratio with fresh MLE12 media containing 8 µg/ml polybrene for 12 hours. Infected MLE12 cells were subcultured for 65 hours prior to sample collection.
|
| Growth protocol |
MLE12 cells were cultured in Hams F12, Corning Cellgro, Manassas, VA; 2% FBS, 0.005mg/ml insulin; 0.01mg/ml transferrin; 30nM sodium selenite; 10nM hydrocortisone;10nM beta-estradiol; 10mM HEPES; and, 2mM L-glutamine, to 60% confluency prior to viral infection.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using Rneasy mini kit (Qiagene)
|
| Label |
biotin
|
| Label protocol |
100ng of total RNA was converted to first-strand cDNA using reverse transcriptase primed by a poly(T) oligomer that incorporated the T7 RNA polymerase promoter. Second-strand cDNA synthesis was followed by in vitro transcription (Affymetrix One-Cycle Target Labeling Kit) for linear amplification and biotinylation of each transcript.
|
| |
|
| Hybridization protocol |
cDNAs were added to Affymetrix hybridization cocktails, heated at 99ºC for 2 min and hybridized for 16 h at 45ºC to GeneChips (Affymetrix Inc., Santa Clara CA). The microarrays were then washed at low (6X SSPE) and high (100mM MES, 0.1M NaCl) stringency and stained with streptavidin-phycoerythrin. Fluorescence was amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain.
|
| Scan protocol |
A confocal scanner was used to collect fluorescence signal after excitation at 570 nm.
|
| Data processing |
Microarray data were analyzed using the Oligo package available at the Bioconductor website (www.bioconductor.org). The raw data were first background-corrected by the Robust Multichip Average (RMA) method and then normalized by an invariant set method. Probesets were considered expressed if at least one sample had a RMA expression value greater than the mean of the negative control probesets, 18,494 probesets were considered expressed. Differential gene expression between the control and mutant mice was analyzed by the Limma package available at the Bioconductor website. P-values obtained from the multiple comparison tests were corrected by false discovery rates. Heatmap displays were created using the freely available MeV package ( http://www.tm4.org/mev/).
|
| |
|
| Submission date |
Nov 14, 2013 |
| Last update date |
Jul 23, 2014 |
| Contact name |
Michael Patrick Morley |
| E-mail(s) |
mmorley@pennmedicine.upenn.edu
|
| Phone |
215-898-2026
|
| Organization name |
Perelman School of Medicine at the University of Pennsylvania
|
| Department |
Penn Cardiovascular Institute
|
| Street address |
3400 Civic Center Blvd, Bldg 421
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL16570 |
| Series (1) |
| GSE52389 |
Long noncoding RNAs are spatially correlated with transcription factors and regulate lung development |
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