Bravo liquid handler

Optimal HTS assay conditions were established based on previous work published elsewhere[15 (link), 22 (link)–24 (link)] and adapted to our system conditions described in Online Resource 1. Cells were exposed to each of the 2,700 medicinally active and structurally diverse compounds contained in the Selleck Bioactive and Selleck FDA-approved libraries (SelleckChem, Houston, TX). Cells were automatically counted, seeded in 384-well plates, and cultured for 24 h. Next, compounds were diluted in 0.5% DMSO (v/v) and transferred into the well plates using an Agilent Bravo Liquid Handler, resulting in a final concentration of 4.8 μM. All plates also included positive (melphalan) and negative (vehicle) controls; melphalan was used based on its potent and reproducible in vitro cytotoxicity[21 (link), 25 (link)]. After 72 h of drug exposure, cell viability was determined[15 (link), 24 (link)]. Compounds that showed > 70% cytotoxic activity at 4.8 μM were considered pharmacologically active against retinoblastoma.

Serum was collected, separated, and stored at −80°C until use. Clinical testing was performed by the Department of Laboratory Medicine at the NIH Clinical Center (Bethesda, MtD) or Mayo Clinic Laboratories (Rochester, MN). PhIPseq services were provided by CDI Labs (Baltimore, MtD). PhIP-Seq was performed as described in Mohan, D., et al. (16 ). Briefly, screens are performed in 1 mL PBS, pH 7.4 containing phage library (~10¹¹ pfu) and 0.2 μL serum (~2 μg IgG). Mock immunoprecipitations containing buffer alone were included as negative control. The mixture is rotated overnight at 4°C to allow antibodies to bind any phage-displayed peptide targets. The next day, 40 μL of a 1:1 Protein A/G coated magnetic bead slurry is added and rotated for an additional 4 hours at 4°C. The beads are then washed three times with TBS, pH 7.4 containing 0.1% NP-40 using a BRAVO liquid handler (Agilent, Santa Clara, CA, USA). The beads are resuspended 20 μL of a Herculase II Fusion Polymerase PCR1 master mix to amplify library inserts. After 20 cycles of PCR, sample-specific barcoding, and the Illumina P5/P7 adapters are then incorporated during a subsequent PCR2 reaction using 2 μL of PCR1 product. After another 20 cycles of PCR, PCR2 amplicons are pooled and sequenced using an Illumina NextSeq to obtain single-end 50 nucleotide reads.

E. coli BW25113 was grown overnight in 3 ml Luria-Bertani (LB) medium and diluted 1/10,000 into fresh LB. 99 µl of cells was added to each well of a 96-well flat-bottom plate (Corning) using a multichannel pipette. Next, 1 µl of a 5 mM stock of each molecule from an FDA-approved drug library supplemented with a natural product library (2,560 molecules total; MicroSource Discovery Systems) was added, in duplicate, using an Agilent Bravo liquid handler. The final screening concentration was 50 µM. Plates were then incubated in sealed plastic bags at 37°C without shaking for 16 hr, and subsequently read at 600 nm using a SpectraMax M3 plate reader (Molecular Devices) to quantify cell growth. Plate data were normalized based on the interquartile mean of each plate.