GSM8455737: MG1655, Stiff PEG, 24 hr, rep 2; Escherichia coli; OTHER
Plasmid DNA from each cell library sample was extracted using the Qiagen QIAPrep Spin Miniprep kit according to the manufacturer's protocol The gRNA spacer region of each plasmid pool sample was amplified using custom primers with variable lengths and sequences at the 5′ end to increase nucleotide diversity while maintaining a total amplicon length of 92 base pairs. Amplification was performed using Q5 polymerase (New England Biolabs, NEB) according to the manufacturer's protocol with 18 cycles. Amplicons were then digested using DpnI (NEB) for 8 hours and the PCR products purified using the GenCatch PCR purification kit according to the manufacturer's protocol. The 20 amplicon samples were then prepared for NGS using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) according to the manufacturer's protocol with 500 ng of starting material and purification of the adaptor-ligated DNA with 0.80X ratio of AMPure XP beads (Beckman Coulter) to for each reaction mixture. NEBNext Multiplex Oligos for Illumina Set 1 or Set 2 primers (NEB) were used to amplify (for 3 cycles) and barcode each sample. DNA concentration was quantified using a Qubit 4 Fluorometer with dsDNA High Sensitivity Quantification assays. All 20 samples were then pooled together in equal mass ratios and sequenced using an Illumina NexSeq500 System with a Mid-150 cycle kit (75 bp X 2 paired end reads) and a 10% PhiX spike-in.