GSM9555794: transposon mutant library pool grown in liquid LB at T2 replicate 2; Bacillus subtilis; Tn-Seq
DNA of Tn-mutant pool was extracted using phenol/chloroform/isoamyl alcohol mixture (PCI, 25:24:1) method, precipitated by sodium acetate and ethanol, and washed twice with 70 % ice-cold ethanol. The purified genomic DNA from Tn library was subjected to MmeI digestion for 2.5 h at 37 °C, then subjected to calf intestinal phosphatase for 1 h at 37 °C. DNA was cleaned up with phenol/chloroform/isoamyl alcohol mixture (PCI, 25:24:1) method, and then ligated with customized illumina R1 adapter. The adapter-ligated DNA junctions were purified using AMPure XP beads. Purified adapter-ligated DNA was used as template for PCR amplification of Illumina sequencing libraries using a universal primer and a specific barcode primer. PCR products were separated on an 8 % non-Urea PAGE gel, and DNA bands of approximately 165 bp were excised. The library DNA was extracted from gel slices and Tn-seq libraries were sequenced using an Illumina NextSeq 550 System with NextSeq 500/550 High Output v2.5 kit (1x 75-bp read length).