Barcode amplicon sequencing of E. coli barcoded deletion library: growth minimal media/condintioned by WT culture from OD0.01; Biological Duplicate 02
Conditioned media was prepared by growing the barcoded _ybcN strain overnight in nutrient-poor media (M9 salts supplemented with 2mM MgSO4, 0.1 mM CaCl2 and 0.5% glucose), supplemented with 20 _g/mL chloramphenicol. When the overnight culture reached an OD600 of 1.2, cells were washed twice in M9 salts and diluted to multiple inoculating densities. Conditioned media was obtained by inoculating the following cell densities:  5x109, 109, 108, and 107 cells/mL, and growing them in nutrient-poor media for 3.5 hours. Cultures were then filtered with a 0.22 micron filter, and nitrogen, phosphorous and carbon sources were replenished by adding x5 concentrated stocks of the M9 salts and glucose. For the screen, 50 _L of the pooled E. coli barcoded deletion library was inoculated into 7 mL of LB media supplemented with 20 _g/mL chloramphenicol and grown over night. This high inoculum was used to maintain a high population size and avoid a sampling bottleneck in the thawing stage. Cells were washed twice in M9 salts and inoculated at a density of 5x106 cells/mL in 7 mL of nutrient-poor media or conditioned media at 37 C. Growth was halted by flash freezing when the cultures reached the OD600 (optical density at 600 nm) range of 0.2-0.3 (typically after about 8 hours of growth). Samples were stored at -80 C for later DNA extraction (Zymo Quick DNA Miniprep Plus Kit, Cat#D4068). A region of ~350 bp around the barcode locus was amplified with custom forward and reverse primers using 2x KAPA HiFi HotStart ReadyMix (Kapa Biosystems, Cat#KK2602) to produce the amplicon libraries for each sample. Sequencing was performed using MiniSeq High Output Reagent Kit, 75-cycles (Illumina, Cat# FC-420-1001) on Illumina MiniSeq device.