Culture medium was exchanged every 7 days and cells were grown in culture for up to 14 days. 24hr prior to experimentation cells were transferred to an hypoxic workstation equilibrated with 4% O2, 5% CO2, and the remaining percentage gas N2. Once cortical astrocytes had reached approximately 90% confluence (75cm2 flask) they were subjected to hypoxia as above, washed with PBS, removed from the flask base with 0.05% trypsin-EDTA (Gibco) and then gently centrifuged (500xg). The cell pellet was then re-suspended in PBS and centrifuged twice more to remove any traces of media. The cell pellet was then triturated in 8-10 volumes of RNAlater (Applied Biosystems), frozen and stored at -80oC.
Extracted molecule
total RNA
Extraction protocol
RNA isolation was carried out using the Qiagen RNeasy Mini Kit for cell extraction (Qiagen, Inc. Valencia CA). The cells were lysed in the proprietary buffer and then centrifuged. The supernatant was transferred to a second tube and centrifuged again to clear any remaining cellular debris. The supernatant was added to 95% ethanol, mixed and added to the proprietary binding columns. The columns were centrifuged, washed several times and the bound RNA was eluted using water. The RNA quality and quantity was checked using an Agilent 2100 Bio-analyzer and the RNA 6000 nano-chips (Agilent Technologies, Palo Alto, CA).
Label
Streptavidin-Cy3 bound to the biotin-labeled cRNA.
Label protocol
standard Illumina protocol using Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791) In short, 0.5g of total RNA was first converted into single-stranded cDNA with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules. The double stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in-vitro transcription reaction where single-stranded RNA (cRNA) was generated and labeled by incorporation of biotin-16-UTP.
Hybridization protocol
standard Illumina protocol. In short, a total of 0.75ug of biotin-labeled cRNA was hybridized at 58 degrees C for 16 hours to Illumina's Sentrix Rat Ref-12,v1 Expression BeadChips (Illumina, San Diego, CA). Each BeadChip has 22,000 well-annotated RefSeq transcripts with approximately 30-fold redundancy. The arrays were washed, blocked and the biotin labeled cRNA was detected by staining with streptavidin-Cy3.
Scan protocol
Arrays were scanned at a resolution of 0.8um using the Beadstation 500 X from Illumina.
Description
Cerebral cortices were removed from 6-8-day-old Wistar rats and placed in ice-cold phosphate-buffered solution (PBS) containing: 8mM NaH2PO4, 2.7mM KCl, 138mM NaCl, and 2.7mM KH2PO4. Multiply-dissected cortices were dispersed into the same buffer containing 0.25 mg/mL trypsin, at 37°C for 15 min. Digestion was halted by the addition of an equal volume of buffer supplemented with 16µg/mL soy bean trypsin inhibitor (type I-S; Sigma, Poole, Dorset, UK), 20U/mL DNase I (EC 3.1.21.1 type II from bovine pancreas; Sigma) and 1.6mM MgSO4. The tissue was then pelleted by centrifugation at 1000xg for 1 min and the supernatant was poured off before resuspending the cell pellet in 6.8mL of buffer solution containing 100µg/mL soy bean trypsin inhibitor, 125U/mL DNase I and 10mM MgSO4. The tissue was subsequently triturated and, after allowing larger pieces of tissue to settle, the cell suspension was pipetted into media (Eagle's minimal essential medium supplemented with 10% fetal calf serum (v/v) and 1% (v/v) penicillin-streptomycin (Gibco, Paisley, UK)). The cell suspension was then aliquoted into 75cm2 flasks. Cells were then maintained in a humidified incubator at 37°C (95% air; 5% CO2). Four to six hours following plating, cells were washed twice with fresh media to remove non-adherent cells. This resulted in a culture of cortical astrocytes, as confirmed by visual inspection the following day and later by glial fibrillary acidic protein immunohistochemistry. Any cortical astrocyte culture that was not homogenous was disposed of and not used in this study. = Rattus norvegicus, cortical astrocytes, cultured at 4% O2.
Data processing
Data was extracted using the Illumina BeadStudio software(v3.4). Any spots at or below the background were filtered out using an Illumina detection value of 0.98 and below. The natural log of all remaining scores were used to find the avg and std of each array and the z-score normalization was calculated and presented below. Z-score = (raw value - avg)/std. Complete data including detection values is included in the supplemental file.
