Hoescht dye

Cell number was evaluated by either direct cell counting (trypan blue exclusion) or high-throughput microscopic counting (HTC) of fixed and stained nuclei. For direct cell counting, at designated time after treatment, media was removed, cells were not rinsed for fear of losing loosely-attached cells, trypsinized, diluted 1:1 with trypan blue and immediate counted on a hemocytometer. For HTC experiments, after designated time period, cells were fixed in 4% PFA either overnight at 4°C or for 30 min at RT. Cells were washed 2x, permeabilized with 0.1% Triton-X in PBS, wash 2x, stained with 50ug/ml Hoescht dye (Sigma cat. no B2261) for 30 min, RT in the dark, then washed 2x and PBST added to each well and scanned by the Cellomics high-throughput microscope at the Duke RNAi Core Facility.

DNA from 1.0-ml EDTA blood vacutainer tubes was extracted (DSP Blood Midi Kit with QIA Symphony DNA extractor, Qiagen). A custom 1.0-ml EDTA blood protocol was run on the QIA Symphony with DNA eluted in 200 μl TE (Qiagen, without azide). DNA from bone marrow aspirates, fresh and frozen tumor tissues, and blood (volume of <1.5 ml or with low white blood cell count) was extracted with the QIAamp Micro silica-based membrane column kit (Qiagen). Specimen input of 1–8 mg of tumor tissue, 50 μl of bone marrow aspirate or 100 μl of blood was extracted on a single QIAamp microcolumn and eluted in 20 μl TE. DNA was extracted from FFPE tissue using the QIAamp DNA FFPE tissue kit (FFPE tumors were included for participants 92, 170, 200, 209, 228 (2 samples), 269, 273 (2 samples), 299, 306, 358, 362, 370, 375, 376, 377, 384 and 400). DNA was quantitated with a Biomek FLX800 fluorimeter using 2 μl DNA, diluted in 198 μl assay B solution containing Hoescht dye (Sigma). A standard curve of 25–450 ng DNA was prepared using commercial genomic DNA (Sigma), and patient DNA was quantified using this curve. Using the SureSelectXT kit, samples were processed using 200 ng DNA. After shearing the DNA (Covaris sonicator), Illumina HiSeq 2500-compatible libraries were generated. Samples were pooled in multiples of six.

Primary neuronal, astrocytes or astrocytic-neuronal co-cultures plated on glass coverslips were washed twice with TBS and fixed at room temperature for 10 min with 100% cold methanol. Cells were permeabilized with 0.1% Triton X-100 in PBS for 15 min and then incubated for 1 h in 5% goat serum to reduce nonspecific background. After an overnight incubation at 4 °C with the primary antibodies: [chicken or mouse anti-glial fibrillary acidic protein (Abcam, ab4674, 1: 200 or Dako, z0334, 1:500), anti-β-III-tubulin, (Abcam, ab18207, 1:500 or Covance, MMS-435P 1:1000), anti-synaptophysin (Abcam, ab106618, 1:500), anti-NeuN (Millipore, MAB377, 1:500)], cells were washed with TBS and incubated with secondary AlexaFluor-conjugated antibodies appropriate for the species (Molecular Probes, 1:500). To visualize cell nuclei, cultures were rinsed and then incubated in 4′,6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI)/antifade (Sigma, 1:1000) diluted in TBS or Hoescht dye (Sigma, 1:5000) for 10 min at room temperature. Cover slips were mounted in FluorSave™ (EMD Millipore) and pictures were taken with a wide-field fluorescence microscope (Leica DMI 4000B microscope using a Leica DFC3000 G camera and the Leica application suite 4.0.0.11706).