Barcode amplicon sequencing of E. coli barcoded deletion library V1: growth condition-M9/Gemcitaine; Replicate 4
To find the ideal screen conditions, a calibration experiment was run using wild-type E. coli (BW25113) with titration of initial bacterial inoculum and gemcitabine doses. Based on the calibration experiment, the genetic screen was performed using 140 M gemcitabine and 1:400 dilution of initial bacterial inoculum (of OD600 of 1). 15 l of pooled barcoded library was inoculated into 25 ml of M9 with chloramphenicol and grown overnight at 37oC, 200 rpm shaking. In the morning, the culture was diluted to OD600 of 1, and then diluted further as 1:400 into 7ml of M9 with or without 140 M gemcitabine (each as four biological replicates). The tubes were incubated at 37oC shaker and OD600 was monitored. Once, OD600 of ~0.6 was reached, the cells were pelleted and genomic DNA was isolated (Zymo Quick DNA miniprep Plus Kit, Cat#D4068). ~350bp region around the genetic barcode was amplified with 2x KAPA HiFi Hotstart ReadyMix (Kapa Biosystems, Cat#KK2602) to prepare the amplicons. Sequencing was performed using NextSeq High Output Reagent Kit, 75-cycles on Illumina MiniNextSeq device.