E-MTAB-3075:gen_t0
Illumina HiSeq 2000 paired end sequencing; Mycoplasma pneumoniae transposon libraries
Mycoplasma pneumoniae transposon libraries
gen_t0
M. pneumoniae M129 strain was transformed with pMT85 and pMTnTetM438 vectors. Those mini-transposon vectors are both derived from Tn4001 and they have different resistance markers under different promoters (24). They are used to avoid bias by vector type in the essentiality study. After transformation, cells were spread on 75 cm2 flasks with 25 ml of Hayflick medium during 4 days. Then, two consecutive passages of the cells (4 days each) were performed and DNA was extracted in each passage by using the Illustrabacteria genomic Kit (GE). Then genomic DNAs were sequenced by HITS approach  Genomic DNAs were sheared to 100 bp DNA fragments by using a Covaris S2 device. Paired-end Illumina libraries were created as described by Bentley et al. (27). After adapters ligation, generated fragments were size selected (between 200 and 400 bp). Enrichment of transposon/chromosomal junction regions was performed by PCR amplification with a 5 - desthiobiotinylated primer specific of the inverted repeat (IR) sequence of transposon and adapter specific PCR PE 2.0 primer. Thermocycler settings were as follows: 30 s, 98°C; 18 cycles of 10 s, 98°C, 30 s, 65°C, 30 s, 72°C; 5 min, 72°C. Fragments between 250 and 300 bp were gel purified and added to Dynabeads® Magnetic Beads (Invitrogen) to capture desthiobiotinylated templates containing transposon insertions. Beads were washed according to the manufacturer’s instructions, and the transposon containing fragments were eluted with 2 mM of biotin. Supernatants were recovered from beads and he resulting transposon libraries were PCR amplified and quantified on an Agilent Bioanalyzer chip (Agilent Technologies). Double-stranded templates were cluster amplified and sequenced on an Illumina GAII.
CRG
E-MTAB-3075:gen_t0
Centre for Genomic Regulation, Barcelona
E-MTAB-3075:gen_t0
Experimental Factor: transformed with pMT85
genotype