Automated Bar-Seq Library Preparation and Pooling v2

Protocol for automated Bar-Seq Library preparation This protocol prepares 96 DNA samples, representing 24 samples from 4 different timepoints, for multiplexed Illumina sequencing. The process starts with two rounds of PCR, each followed by a bead-based cleanup. The first round of PCR attaches primers that serve as tags to identify the timepoint and sample (e.g., time-points 1-4 in the Pooled, Growth-Based Assay Protocol). The second round of PCR attaches flow-cell adapters required for Illumina sequencing. Following the PCR and cleanup steps, the protocol outlines a procedure for pooling all samples together to ensure balanced representation during sequencing. This process starts with quantifying each sample's DNA concentration and then diluting and pooling together samples from the same timepoint. The pooled samples' concentrations are then measured, and a small quantity of each timepoint sample is run on a gel to ensure the cleanup process was successful. The final step is to dilute and pool of all timepoint samples into one multiplexed tube for subsequent Illumina sequencing. NOTES: Before implementing this protocol, prepare the magnetic bead suspension following the Preparation of Sera-mag SpeedBeads protocol. This protocol should be implemented after the Automation Protocol for Plasmid DNA Extraction fromE. coli protocol A fragment analyzer can also be used instead of a gel to determine if the PCR clean-up process was a success