TNPseq of Corynebacterium glutamicum CR101: ISCg1-mediated insertion of neo into ML103
For sequencing of complete mutant libraries, all colonies were pooled before genomic DNA was isolated. Subsequently, the gDNA was fragmented to  10,000 bp fragments using a Covaris g-TUBE. The ends of the fragments blunted and an A-overhang was added with the NEBNext Ultra II End Repair/dA-Tailing Module. The A-overhang is then used for ligation of dsDNA adapters derived from annealing the oligos D7 and D7 top carrying part of the TruSeq D7 sequence. In a first round of PCR using Phusion HighFidelity DNA polymerase, fragments are amplified between a biotinylated primer tnp o, binding in the kanamycin resistance marker about 500 nt upstream of the inverted repeat (IR), and the TruSeq D7 primer. After amplification, fragments were purified with Dynabeads M-270 Streptavidin binding the biotinylated fragment ends. During washing of the trapped DNA on a magnet rag, 0,1 mM NaOH was applied to retain ssDNA of the amplified region. This is followed by a half-nested second round of PCR with LongAmp Taq DNA Polymerase. Here, primer D7 is paired with a phosphorylated nested primer tnp i, located 150 bp from the 3-end of the kanamycin resistance cassette. After purification, the DNA was used in the adapter ligation step of the Ligation Sequencing Kit SQK-LSK109 (Oxford Nanopore Technologies) and sequenced on an R9.4.1 flowcell on a GridION.