|
| Status |
Public on Jun 01, 2017 |
| Title |
v-Hras transduced mouse epidermal keratinocytes + control ShRNA lentivirus biol replicate 1 |
| Sample type |
RNA |
| |
|
| Source name |
primary mouse epidermal keratinocyte
|
| Organism |
Mus musculus |
| Characteristics |
protocol: yes v-Hras retrovirus
shRNA: scrambled ShRNA
strain: FVB/n
|
| Growth protocol |
Primary mouse keratinocytes were isolated from newborn FVB/n mice according to standard protocols and cultured in EMEM media with 8% chelexed serum, antibiotics and 0.05 mM calcium chloride. Keratinocytes were co-infected with a high titer v-Hras retrovirus on day 2 of culture and either scrambled control ShRNA, Ire1a ShRNA or Xbp1 ShRNA lentiviruses and after 48 hrs selected for 2 days in puromycin. Total RNA was isolated from triplicate cultures on day 5 after virus transduction. Primary keratinocytes without v-HRas were transduced with the scrambled control ShRNA lentivirus, selected in puromycin and Total RNA isolated from these cells at the same total time in culture as virus transduced keratinocytes.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated keratinocyte cultures using Ribozol on day xx after lentiviral transduction and selection. RNA quality assesed using Agilent Bioanalyzer.
|
| Label |
biotin
|
| Label protocol |
cDNA was generated from 100 ng of total RNA from each sample using the Ambion WT Expression Kit according to the manufacturer’s protocol. The sense strand cDNA was fragmented and labelled using the Affymetrix GeneChip WT Terminal Labeling Kit according to the manufacturer’s instructions
|
| |
|
| Hybridization protocol |
Samples were hybridized according to manufacturer's instructions using the GeneChip Hybridization Wash and Stain Kit and Affymetrix GeneChip Hybridization Oven 640. Arrays were washed and stained using the Affymetrix GeneChip Fluidics Station 450 according to the manufacturer’s protocol.
|
| Scan protocol |
Affymetrix Gene ChIP Scanner 3000 7G
|
| Data processing |
Data were processed using ArrayStar 11 software. RMA background correction was applied to probe intensities. Gene level expression were summarized from the corrected intensities and quantile normalized.
MoGene-2_0-st.pgf
MoGene-2_0-st-v1.na33.mm10.transcript.csv
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| |
|
| Submission date |
Jul 14, 2015 |
| Last update date |
Jun 01, 2017 |
| Contact name |
Adam B. Glick |
| E-mail(s) |
abg11@psu.edu
|
| Organization name |
The Pennsylvania State University
|
| Department |
Veterinary and Biomedical Sciences
|
| Street address |
101 Life Sciences Bld
|
| City |
University Park |
| State/province |
PA |
| ZIP/Postal code |
16802 |
| Country |
USA |
| |
|
| Platform ID |
GPL16570 |
| Series (1) |
| GSE70899 |
Effect of IRE1a and XBP1 knockdown on gene expression in primary mouse keratinocytes expressing an HRas oncogene |
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