title: GSM6033851: NaCl; Escherichia coli str. K-12 substr. MG1655; OTHER
library_construction_protocol: Mutations on chromosomes were consistent with those carried by the CREATE plasmids. Firstly, the CREATE plasmids of tolerant strains were extracted separately. Following extraction, the CREATE plasmids were used as templates for PCR using Illumina-compatible primers designed with Golay barcodes . CREATE library fragments were amplified in 16 cycles of PCR. At the first PCR step, each experimental sample was ligated with a unique barcode used to track the mutations. After that, PCR products were confirmed by electrophoresis and purified using an Agarose Gel DNA Extraction Kit. Deep sequencing was performed according to the Illumina protocol for amplicon sequencing. using global regulator libraries based on the CRISPR-enabled trackable genome engineering (CREATE) method to engineer tolerance against multiple inhibitors in Escherichia coli. Deep mutagenesis libraries were rationally designed, constructed, and screened to target 34,340 mutations across 23 global regulators.These libraries were divided into G1, G2, G3, G4 and G5 sub-groups according to different gene functions (such as active sites, DNA binding sites, and predicted functional sites) . Illumina protocol for amplicon sequencing