|
| Status |
Public on Oct 12, 2011 |
| Title |
Control_24h_Replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
HEKn Control 24h
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Human keratinocytes (HEKn)
treatment agent: control (untreated)
timepoint: 24 hours
|
| Treatment protocol |
HEKn cells were incubated in the absence (n = 3) and presence (n = 5) of 500 ng/ml recombinant human IL-17C (in-house) for 3 and 24 hours.
|
| Growth protocol |
HEKn keratinocytes (Invitrogen) were cultured in EpiLife medium (Invitrogen) supplemented with Human Keratinocyte Growth Supplement (HKGS, Invitrogen). For experiments, HEKn were grown to confluence in 12 well plates treated with Coating Matrix (Invitrogen).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Whole RNA was isolated via RNeasy kit (Qiagen).
|
| Label |
Cy5
|
| Label protocol |
1 ug of total RNA was converted into double-stranded cDNA using a T7 Promoter Primer and MMLV-RT (Agilent, Low RNA Input Fluorescent Linear Amplification Kit, Product # 5184-3523). After cDNA synthesis, cRNA was synthesized using T7 RNA polymerase, which simultaneously incorporated cyanine 3- or cyanine 5- labeled CTP. The labeled cRNA was purified on an affinity resin column (RNeasy Mini Kits, Qiagen). The amount of labeled cRNA was determined by measuring absorbance at 260 nm and using the convention that 1 OD at 260 nm corresponds to 40 ug/ml of RNA. Incorporation of dye was determined by measuring the sample using the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE) which measured the absorbance of cyanine 3- and cyanine 5- labeled CTP. Eight hundred twenty-five nanograms of cyanine 3-labeled Universal Human Reference cRNA (Stratagene, La Jolla, CA, Product # 740000) and 825 ng of cyanine 5-labeled cRNA was fragmented by incubating at 60°C for 30 minutes in fragmentation buffer (Agilent In situ Hybridizatyion kit-plus, Product # 5184-3568). Fragmentation was terminated by adding hybridization buffer containing LiCl and lithium lauryl sulfate.
|
| |
|
| Channel 2 |
| Source name |
Stratagene Universal Human Reference RNA
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: reference
|
| Treatment protocol |
HEKn cells were incubated in the absence (n = 3) and presence (n = 5) of 500 ng/ml recombinant human IL-17C (in-house) for 3 and 24 hours.
|
| Growth protocol |
HEKn keratinocytes (Invitrogen) were cultured in EpiLife medium (Invitrogen) supplemented with Human Keratinocyte Growth Supplement (HKGS, Invitrogen). For experiments, HEKn were grown to confluence in 12 well plates treated with Coating Matrix (Invitrogen).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Whole RNA was isolated via RNeasy kit (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
1 ug of total RNA was converted into double-stranded cDNA using a T7 Promoter Primer and MMLV-RT (Agilent, Low RNA Input Fluorescent Linear Amplification Kit, Product # 5184-3523). After cDNA synthesis, cRNA was synthesized using T7 RNA polymerase, which simultaneously incorporated cyanine 3- or cyanine 5- labeled CTP. The labeled cRNA was purified on an affinity resin column (RNeasy Mini Kits, Qiagen). The amount of labeled cRNA was determined by measuring absorbance at 260 nm and using the convention that 1 OD at 260 nm corresponds to 40 ug/ml of RNA. Incorporation of dye was determined by measuring the sample using the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE) which measured the absorbance of cyanine 3- and cyanine 5- labeled CTP. Eight hundred twenty-five nanograms of cyanine 3-labeled Universal Human Reference cRNA (Stratagene, La Jolla, CA, Product # 740000) and 825 ng of cyanine 5-labeled cRNA was fragmented by incubating at 60°C for 30 minutes in fragmentation buffer (Agilent In situ Hybridizatyion kit-plus, Product # 5184-3568). Fragmentation was terminated by adding hybridization buffer containing LiCl and lithium lauryl sulfate.
|
| |
|
| |
| Hybridization protocol |
Hybridization and washing were performed using a fully automated hybridization apparatus, the Tecan HS4800 Pro Hybridization Station (TECAN U.S., Research Triangle Park, NC).
|
| Scan protocol |
Arrays were scanned in the Agilent G2505C model Scanner using Agilent Scan Control Software (version A.8.4.1).
|
| Description |
Biological replicate 2 of 3. HEKn cells, untreated, harvested after 24 hours.
SAM627591
|
| Data processing |
Expression signals were calculated using the Agilent Feature Extraction software (version 10.7.3.1 Agilent Technologies, Santa Clara, CA).
|
| |
|
| Submission date |
Oct 04, 2011 |
| Last update date |
Oct 12, 2011 |
| Contact name |
Alex Abbas |
| E-mail(s) |
[email protected]
|
| Organization name |
Genentech, Inc.
|
| Department |
Bioinformatics
|
| Street address |
1 DNA Way
|
| City |
South San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94080 |
| Country |
USA |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE32620 |
Human keratinocytes stimulated with IL-17C |
|
