Tn Seq of Clostridium autoethanogenum
Sequencing library preparation was performed as an amplicon library using a splinkerette adapter 49. Genomic DNA was fragmented to an average of 400 bp using a covaris sonicator followed by bead purification using NEB sample preparation beads at a ratio of 1.5X beads to sample. Fragmented DNA was end repaired and A-tailed using the NEB Ultra II library preparation kit. Splinkerette adapters were ligated onto the end of A-tailed fragments with reagents from the Ultra II library preparation kit. A 1X bead purification was performed before an I-SceI digest step to cleave plasmid DNA between the library primer and P7 primer. Another 1X bead purification was performed before PCR amplification of the transposon junctions using KAPA HiFi polymerase. An initial denaturing step of 95C for two min was followed by 20 rounds of 95C for 20 sec, 61C for 30 sec then 72C for 30 sec before a final extension of two min at 72C was performed.