DNA-seq of E. coli: BYR library
Plasmid DNA was isolated from E. coli cell pellets using ZymoPURE II Plasmid Maxiprep Kit (Zymo Research, D4203) per manufacturer's instructions. Amplicon pools containing the TSS and barcode regions were generated by Micellula DNA Emulsion & Purification (ePCR) Kit (EURx/CHIMERx, 3600) per manufacturer's instructions. To minimize amplification bias, each sample was amplified in a 15-cycle ePCR reaction, purified and subject to an additional 10-cycle scale-up ePCR reaction. To create the necessary sequence diversity for sequencing, 18-25 bp and 1-7 bp "stuffer" sequences were added to 5'- and 3'-end, respectively, during amplicon preparation. The amplified amplicon pools were subject to Illumina NovaSeq 6000 (150 PE) sequencing, and on average 20 M paired-end reads were obtained from each replicate of a sample, with high reproducibility and minimal perturbation of the variant distribution with each library.