title: Illumina HiSeq 2500 paired end sequencing; Transposon sequencing to identify random insertions of lox sites in Mycoplasma pneumoniae
design: Transposon sequencing to identify random insertions of lox sites in Mycoplasma pneumoniae
library_construction_protocol: WT M129 cells were transformed with 1 pMole of the plasmid pMTnLox66Cm, via electroporation. 125 µl of transformed cells were then inoculated into a T75 cm2 culture flask containing 20 ml Hayflick and supplemented with chloramphenicol. The culture was grown to mid-log phase then cells were isolated via centrifugation (Sample R1B1).   Aliquots of 50 µl from the previous homogenised cells were mixed with a pre-chilled 30 µl EB solution containing the 1 pMole of pMTnLox71Tc plasmid DNA, and transformed as in the first round. Cells were recovered in the same manner, and 125µl were inoculated into a T75 cm2 flask containing 20 ml Hayflick media supplemented with 2 µg/ml tetracycline and 20 µg/ml chloramphenicol.  Genomic DNA was isolated via the MasterPure™ genomic DNA purification kit. Between 10 and 500 ng genomic DNA were fragmented to 200-300 bp using a Covaris S2 instrument. End repair and adaptor ligation was performed using the NEBNext Ultra DNA Library Prep kit for Illumina.