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Expression data from sorted epithelial CD34+ expressing cells from DMBA/TPA induced skin tumors

ABSTRACT: Transcriptional profile of control and VEGF overexpressing FACS-isolated CD34+ Cancer stem cells from DMBA/TPA induced skin tumours

ORGANISM(S): Mus musculus

PROVIDER: GSE31465 | GEO | 2011/09/07

SECONDARY ACCESSION(S): PRJNA145661

REPOSITORIES: GEO

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Expression data from sorted epithelial CD34+ expressing cells from DMBA/TPA induced skin tumors

Project description:Transcriptional profile of control and VEGF overexpressing FACS-isolated CD34+ Cancer stem cells from DMBA/TPA induced skin tumours Tumours were digested in collagenase I (Sigma) for 2 hours at 37°C on a rocking plate. Collagenase I activity was blocked by addition of EDTA (5mM) and then rinced in PBS supplemented with 2%FCS. After tumour digestion, cells were first incubated in PBS complemented with 30% FCS to block Fc receptors for 15 min at room temperature. Immunostaining was performed using biotin-conjugated anti-CD34 (clone RAM34; BD Pharmingen), FITC-conjugated anti-α6-integrin (clone GoH3; BD Pharmingen), PE-conjugated anti-CD45 (clone 30F11, eBiosciences), PE-conjugated anti CD31 (clone MEC13.3; BD Pharmingen), PE-conjugated anti-CD140a (clone APA5; eBiosciences), APC-Cy7-conjugated anti-Epcam (clone G8.8; Biolegend) by incubation for 30min on ice. Cells were washed and stained using APC-conjugated Streptavidin (BD Pharmingen) for 20min on ice. Living tumour cells were selected by forward scatter, side scatter and by Hoechst dye exclusion. Fluorescence-activated cell sorting analysis was performed using FACSAria and FACSDiva software (BD Biosciences). Sorted cells were collected in the lysis buffer provided by the manufacturer (Microprep kit, RNeasy, Stratagene) and RNA extraction was performed according to the manufacturer's protocol. Total RNA was analysed using Mouse whole genome 430 2.0 array from Affymetrix at the VIB microarray facility (KU – Leuven, Belgium). We analyzed CD34+ TECs isolated by FACS from a K14CreER:Rosa-VEGF-164 and control mice. Analysis of the microarray was perfored by the VIB microarray facility (KU – Leuven, Belgium).

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