GSM8279339: plasmid DNA fragments, amplified, library before sorting in the second round; Escherichia coli str. K-12 substr. MG1655; OTHER
Plasmid extraction was performed using the TIANprep Mini Plasmid Kit (Tiangen, China). The plasmids were used as templates for PCR to amplify the N20 region of library sgRNAs. The PCR fragment was purified using the TIANquick Midi purification Kit (Tiangen, China). For each sample, the purified PCR products were treated with End Prep Enzyme Mix for end repairing, 5' phosphorylation, and dA-tailing in one reaction, followed by a T-A ligation to add adaptors to both ends. Adaptor-ligated DNA was then performed using DNA Clean Beads. A second PCR reaction was carried out with P5 and P7 primers carrying sequences which can anneal with flowcell to perform bridge PCR and index allowing for multiplexing. The products were purified by beads and then qualified. The final libraries were sequenced via the Illumina NovaSeq 6000 platform using a 2 × 150 paired-end configuration.