cell line: SF8628 age: year 3 tissue: Pons gender: female
Growth protocol
Primary pediatric human glioma cells, designated with “SF”, were obtained from surgical biopsy of tumor from patients admitted to UCSF medical center, and in accord with an institutionally approved protocol. Establishment of glioma cell cultures, from surgical specimens, and tumor cell modification for expression of firefly luciferase, for in vivo bioluminescence imaging, have been described inHashizume R et al.,2012 (http://www.ncbi.nlm.nih.gov/pubmed/22983601)
Extracted molecule
total RNA
Extraction protocol
Briefly, 1×10^6 cells, treated with vehicle or 6 μM GSKJ4 for 24 and 72 hours, were harvested, washed with PBS and fixed with 1% formaldehyde for 5 minutes at room temperature. Cells were then quenched with 125 mM glycine for 5 minutes. After wash, cell extracts were prepared using lysis buffer (50 mM HEPES/KOH at pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton-X100, 0.1% Na-deoxycholate, 1 mM PMSF, 1 mM Pefoblock, 1 mM benzamidine, 1 mg/ml bacitracin) on ice for 10 minutes.
Label
Cy3
Label protocol
RNAs from SF8628 K27M DIPG cells, either untreated or treated with GSKJ4 for 24 and 72 hours, were extracted then labeled (Cy3 for treated and Cy5 for untreated).
cell line: SF8628 age: year 3 tissue: Pons gender: female
Growth protocol
Primary pediatric human glioma cells, designated with “SF”, were obtained from surgical biopsy of tumor from patients admitted to UCSF medical center, and in accord with an institutionally approved protocol. Establishment of glioma cell cultures, from surgical specimens, and tumor cell modification for expression of firefly luciferase, for in vivo bioluminescence imaging, have been described inHashizume R et al.,2012 (http://www.ncbi.nlm.nih.gov/pubmed/22983601)
Extracted molecule
total RNA
Extraction protocol
Briefly, 1×10^6 cells, treated with vehicle or 6 μM GSKJ4 for 24 and 72 hours, were harvested, washed with PBS and fixed with 1% formaldehyde for 5 minutes at room temperature. Cells were then quenched with 125 mM glycine for 5 minutes. After wash, cell extracts were prepared using lysis buffer (50 mM HEPES/KOH at pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton-X100, 0.1% Na-deoxycholate, 1 mM PMSF, 1 mM Pefoblock, 1 mM benzamidine, 1 mg/ml bacitracin) on ice for 10 minutes.
Label
Cy5
Label protocol
RNAs from SF8628 K27M DIPG cells, either untreated or treated with GSKJ4 for 24 and 72 hours, were extracted then labeled (Cy3 for treated and Cy5 for untreated).
Hybridization protocol
One hundred ng quantities of corresponding treated – untreated pairs were co-hybridized to SurePrint G3 Human Gene Expression 8x60K Microarrays (Agilent Technologies), in duplicate, for each length of treatment, for 17 hours at 65 °C
Scan protocol
Scanned on an Agilent microarray scanner (model G2505C) Images were quantified using Agilent’s Feature Extraction 10.5.1 software.
Agilent’s Feature Extraction 10.5.1 software with Agilent’s Two Color technology option was used for thresholding of signal values to five, ratio computing (cy3/cy5), and log transformation
