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Beads were read on a Bio-Plex Suspension Array System and the data were analysed using Bio-Plex Manager software (V.4.0).
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Streptavidin-phycoerythrin was then added, and the beads were read using the Luminex 200 system (Luminex Corporation).
After the addition of AlphaScreen beads (PerkinElmer) and detection antibody (Cell Signaling Technology, #4735) followed by overnight incubation at room temperature, the plates were read on an Envision 2101 (PerkinElmer).
Finally, the plate was read on Luminex set for specified bead regions with the following parameters: 100-μl sample size; 5000 25,000 DD gate; 45-sec timeout; and 100 bead events per bead region.
Target-specific detection probes are then coupled to carboxyl groups on a bead-based substrate, and probe fluorescence is read using a bead-based detection platform.
The fluorescent signals are read using the Illumina Bead Array Reader software.
The qPCR pool was sequenced on lane 1, and the Bioanalyzer pool was sequenced on lane 2. A total of two million beads were loaded on each lane resulting in 883,053 reads that passed all GS FLX quality control filters.
Enriched beads were sequenced on an ABi SOLiD 4 analyzer according to the manufacturer's instructions to generate 50 bp reads in color space.
Microcarrier beads were scattered on the strip.
Additional Templated beads were deposited on slides using the Bead Deposition kit from Applied Biosystems.
The resulting beads were then deposited on the SOLiD flow cell and subjected to massively parallel 50 bp single-read sequencing on a Life Technologies SOLiD sequencer.
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