DNA-seq of LasR protein library from FACS bin 2 treated by 1 micromolar 3OC12-HSL, rep A
The LasR variable region for each sample was amplified (113 bp). PCR reactions were performed using KAPA HiFi HotStart DNA polymerase for 22 cycles. The PCR products were purified and eluted in 0.1 X TE buffer. The amplicon library (235 bp), containing adaptor sequences for Illumina NextSeq500, a unique barcode for each sample, and the PCR product, was created using the NEBNext Ultra II DNA library prep kit for Illumina (NEB #7103S) with index primer set 1, index 2 (NEB #7335S) according to the manufacturers protocol with minor modifications. 600 ng of PCR product was used as the starting material, and clean-up of adaptor-ligated DNA (with 0.8X resuspended beads) instead of size selection was performed because the length of PCR amplicon was uniform (113 bp). 4 PCR cycles were chosen for PCR enrichment of adaptor-ligated DNA. The resulting amplicon libraries were purified using NEBNext Sample Purification Beads in the kit and a magnetic stand (EpiMag HT (96-Well) Magnetic Separator, Epigentek group INC.).