High-Throughput Bioscience Center (HTBC)

1. High-Throughput Bioscience Center (HTBC) • Access to rapidly test thousands of molecules in various bioassays. • Small Molecule Compound library—114,000 diverse compounds (ChemDiv, Chembridge, Specs, ChemDiv Kinase) • Drug Repurposing Libraries—8,770compounds of known modulators/FDA approved Drugs (LOPAC, NIHCC, NCI DTP, Micorsource, Biomol ICCB and FDA) • Small interfering RNAs (siRNAs)—Wholegenomes targeting 21,000 genes, pools of 4 siRNAs/gene, Human library from Dharmacon, and Mouse library from Qiagen • cDNAs—15,000 genes,E. coli stocks Human ORFeome in Gateway vectors from Open Biosystems • High-Throughput Assays (384-well plates) • In vitro targets--Enzymes, Protein-protein interactions, Ligand-receptor binding, Protein-DNA binding, etc. (10-15,000 compounds/day) • In vivo cell based-spectrophotometric--Reporter gene-luciferase, proliferation, synthetic lethality, etc. (15-30,000 compounds per week) • High-Content Imaging--Mitosis, protein translocation/location/expression, cellular morphology, apoptosis, (5-10,000 compounds per week) • Fully automated integrated system: • 8, 96 or 384 tip pipettors/pin tools (CaliperLS SciClone ALH 3000) • Robotic arms/Stackers for moving microplates (Zymark Twister II) • Robotic 198 plate CO2 Incubator • Integrated plate washer/reagent dispenser (Bio-tek EL406) and reagent dispenser (Multidrop) • Bar Code reader • Microplate reader (Molecular Devices AnalystGT, Tecan Infinite M1000)

2. siRNA Screening at the HTBC • siARRAY whole Human Genome siRNA library from ThermoFisher (formerly Dharmacon) • 80 gene targets per plate • 4 siRNA oligos per gene--pooled in each well • 267 X 96 well plates targeting 21,122 genes • Library subdivided in: • 10 Kinase (779 genes, can be tested separately) • 7 GPCR (517 genes) • 76 Druggable genome (6022 genes) • 174 Rest of genome including XMs (13,804 genes) • All individual siRNA oligos available for retests of hits • 4 individual oligos per gene • High-Throughput siRNA assays performed in 96- or 384-well plates (384-well preferred) • Mouse siRNA library from Qiagen formatted in 384-well plates. • Agilent system (96/384 well) • 6 stackers • Bravo 96 or 384 tip pipetting head- 9 plate positions • Thermal Plate Sealer • Wellmate Reagent Dispenser (96/384 well) • ELx405 Plate Washer (96/384 well)

3. High-Content Cellular Imaging • ImageXpress Micro (Molecular Devices) • Fully automated inverted epifluorescence microscope scans 6- to 384-well plates and records multiple fluorescent images • Live cell option-Temp, CO2, humidity • Xenon Lamp/filter based • Complete spectral range with 15 filter set combinations • Up to four color imaging/any fluorophore • Phase Contrast and Brightfield • Robotic Arm Integration • 3 stackers for fixed plates • Automated incubator for imaging up to 42 live-cell plates • Image Analysis Software • MetaXpress 5 with 10 Analysis modules • 2 analysis computers • 3rd party/custom/ Matlab, Cell Profiler • Completed fully automated whole genome siRNA High Content Screen • 200 X 384-well plates imaged, 1 site, 4 colors in 10 weeks; 307,000 images! • More Info: http://htbc.stanford.edu David E. Solow-Cordero, Ph.D. Director, HTBC, CCSR-0133 desolow@stanford.edu