title: Illumina HiSeq 2500 paired end sequencing; DNA sequencing to validate random deletions in Mycoplasma pneumoniae
design: DNA sequencing to validate random deletions in Mycoplasma pneumoniae
library_construction_protocol: WT M129 cells were transformed according to the protocol outlined by Hedreyda et al (Hedreyda et al., 1993). Cells were grown to mid-log phase, identified by the Hayflick media changing from red to orange. The media was decanted and the flask was washed 3x with 10 ml chilled electroporation buffer (EB: 8mM HEPES, 272nM sucrose, pH 7.4). Cells were scraped into 500 µl chilled EB and homogenised via 10x passages through a 25-gauge syringe needle. Aliquots of 50 µl of the homogenised cells were mixed with a pre-chilled 30 µl EB solution containing the 1 pMole of pMTnLox66Cm plasmid DNA. Samples were then kept on ice for 15 mins. Electroporation was done using a Bio-Rad Gene Pulser set to 1250 V, 25 µF and 100Ω. After electroporation, cells were incubated on ice for 15 mins, then recovered into a total of 500 µl Hayflick media and incubated at 37°C for 4 hours. 125 µl of transformed cells were then inoculated into T75 cm2 culture flasks containing 20 ml Hayflick and supplemented with 20 µg/ml chloramphenicol. The transformed cells were grown to mid-log phase, then isolated via pelleting. To ensure the recovery of planktonic cells, the media was transferred to a 50 ml Falcon tube. The flask was then scraped into 500 µl Hayflick, which was added to the Falcon tube with the media. The sample was centrifuged at 10,000 RPM at 4°C for 10 mins to pellet the cells. The supernatant was discarded and the cells resuspended in 500 µl chilled EB. The cells were homogenised via 10x passages through a 25-gauge syringe needle. Aliquots of 50 µl of the homogenised cells were mixed with a pre-chilled 30 µl EB solution containing the 1 pMole of pMTnLox71Tc plasmid DNA, and transformed using the previously described settings. 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. The transformed cells were again grown to mid-log phase and isolated via the centrifugation method described above. Aliquots of 50 µl of the homogenised cells were mixed with a pre-chilled 30 µl EB solution containing the 1 pMole of pMTnCreGm plasmid DNA, and transformed using the previously described settings. Cells were recovered in the same manner, and 125 µl were inoculated into a T75 cm2 flask containing 20 ml Hayflick media supplemented with 200 µg/ml gentamicin.  isolated cells that survived transformation with the pMTnCreGm, 100µl was isolated, and had genetic material isolated via the MasterPure™ genomic DNA purification kit (Lucigen, Cat. No. MC85200).  Genomic DNA was fragmented to 300 bp via Covaris sonication. 5' phosphorylation was undertaken to allow for adapter binding, then 3' overhangs were filled to create blunt ends. These were then ligated using T4 ligase to create circular fragments, and linear DNA was removed via digest with exonuclease I and lambda exonuclease. Circular DNA was then denatured and amplified using a primer mix containing LE-RE_Lox72, loxloop-6, loxloop-7 and loxloop-8 and a phi29 polymerase to amplify DNA containing a deletion scar. This amplified product was then fragmented again using Covaris to 300 bp and NEBNext Adaptor for Illumina were annealed to the linearised DNA.