Low-iron, replicate 1, timepoint 3, tile 3
A plasmid library of EccD3 mutant variants in the pMV_306 vector was electroporated and expressed in a M. smegmatis fxbA/eccD3 double knockout strain for a selection screen in the absence and presence of iron. Genomic DNA (gDNA) was isolated from cells using the Zymo Quick-DNA Microprep Plus kit. The library was PCR amplified to produce three tiles using a third of all gDNA for each PCR using TakaraBio Primestar GXL, with a final forward and reverse primer concentration of 0.3uM using the following parameters: initial denaturation at 98C for 30s, 24x cycles of denaturation at 98C for 10 sec, annealing at 60C for 15 sec, extension at 68C for 30 sec, and a final extension at 68C for 1 min. PCR reactions were concentrated using Zymo DNA Clean and Concentrator-25 kits. Samples were run on 1% 1x TBE gels to confirm amplicons were successfully amplified. Libraries were indexed using the IDT for Illumina-TruSeq DNA and RNA UD Indexes. The lengths of the Indexed libraries were quantified using the Agilent TapeStation with HS D5000 screen tape and reagents (Agilent). DNA concentrations were quantified using the Qubit dsDNA HS assay (Invitrogen). All samples were normalized to 4nM and pooled and then paired-end sequenced (V3) on a MiSeq.