|
| Status |
Public on Aug 08, 2015 |
| Title |
LightMelanocytes_24h_KCM-_rep5 |
| Sample type |
RNA |
| |
|
| Source name |
LightMelanocytes_24h_KCM-_rep5
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: skin
cell type: epidermal keratinocytes
pigmentation level: light
media: KCM-
hours post irradiation: 24
|
| Treatment protocol |
Keratinocyte supernatants were harvested from both non-irradiated (hereinafter KCM-) and irradiated keratinocytes (24 hours after treatment) (hereinafter KCM+) and kept frozen at -80ºC until use. Subconfluent melanocyte cultures were cultivated in Medium 254 supplemented with HMGS and KCM+ or KCM- Medium in a proportion 1:1. The following day they were irradiated with 75 mJ/cm2 of UVB, and harvested at 6, 12 and 24 hours post irradiation. We used non-irradiated control cultures that were covered by aluminium foil during irradiation (Fig 1).
|
| Growth protocol |
Human epidermal keratinocytes were cultured in EpiLife Medium supplemented with human keratinocyte growth supplement (HKGS). Human epidermal melanocytes were cultivated in Medium 254 supplemented with 1% human melanocyte growth supplement (HMGS). All the cell lines were maintained in an incubator under an atmosphere of 5% CO2 at 37ºC. Media were refreshed every two days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction kit from Ambion
|
| Label |
Cy3
|
| Label protocol |
100 ng RNA were labelled with the Low input Quick Amp Labeling kit, one color (Agilent)
|
| |
|
| Hybridization protocol |
The hybridization step was performed using SureHyb hybridization chamber (Agilent Technologies) and 600 ng of labeled cRNA samples, for 17 hours at 65ºC and 10,000 rpms in an hybridization oven. Microarrays were stabilized with ozone-barrier slide covers (Agilent).
|
| Scan protocol |
Image processing of the microarrays was performed by using Agilent Feature Extraction software v10.7.3.1.
|
| Data processing |
Raw data were processed with GeneSpring GX software v11.5.1 (Agilent). Feature extraction flags were transformed as follows: if feature was not positive and significant, not uniform, not well above background or was a population outlier: compromised; if feature was saturated: not detected. our data were subjected to a DDHF (Data-Driven Haar-Fisz) transformation for variance stabilization with the R package DDHFm (Motakis et al, 2006). Data were transformed to log base 2 and normalized following the quantile method (Bolstad et al, 2003). Flag spot information in data files was used to filter probe sets. Entities in which more than 50% of samples in 1 out of any 7 conditions (0h, 6h KCM-, 12h KCM-, 24h KCM, 6h KCM+, 12h KCM+ and 24h KCM+) had “detected” flags were maintained for the analysis.
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| |
|
| Submission date |
Jun 25, 2015 |
| Last update date |
Aug 09, 2015 |
| Contact name |
Saioa Lopez |
| E-mail(s) |
saioa.lopez@ucl.ac.uk
|
| Organization name |
UCL
|
| Street address |
Gower Street
|
| City |
London |
| ZIP/Postal code |
WC1E 6BT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL14550 |
| Series (1) |
| GSE70280 |
Comparison of the Transcriptional Profiles of Melanocytes from Dark and Light Skinned Individuals under Basal Conditions and Following Ultraviolet-B Irradiation |
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