ERX2933063 E-MTAB-7425:agalactiae_wt_SynMyco_R1_p ERP112307 PRJEB29946 SynMyco transposon: engineering transposon vectors for efficient transformation of minimal genomes ERS2914955 SAMEA5130573 agalactiae_wt_SynMyco_R1_p WGA GENOMIC PCR M. agalactiae was transformed using the pMTnGm-SynMyco transposon. Two hours after the transformation, 4/5 of the culture was inoculated into 10 mL SP4 + 0.5% sodium pyruvate (Sigma-Aldrich P8574-5G) + 100 µg/mL gentamicinl. After 24 hours, the culture was collected in 500 µl of SP4. A volume of 8 µl of this culture was then inoculated into 10 mL of fresh SP4 + 0.5% sodium pyruvate (Sigma-Aldrich P8574-5G) + 100 µg/mL gentamicin. After 24 hours, the culture was collected in 500 µl of SP4 (pass 2). A volume of 8 µl of this culture was then inoculated into 0 mL of fresh SP4 + 0.5% sodium pyruvate (Sigma-Aldrich P8574-5G) + 100 µg/mL gentamicin. After 24 hours the culture was collected in 500 µl SP4. From this volume, 350 µl was taken to extract genomic DNA. The number of divisions expected was 20. The genomic DNA was isolated using the Masterpure Complete DNA & RNA Purification Kit (MC85200, Lucigen) following the protocol described by the manufacturer. 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. 250 0 F Application Read Forward 1 1 R Application Read Reverse 126 Illumina HiSeq 2000 Experimental Factor: genotype pMTnGm-SynMyco-Tn4001 ERX2933064 E-MTAB-7425:agalactiae_wt_SynMyco_R2_p ERP112307 PRJEB29946 SynMyco transposon: engineering transposon vectors for efficient transformation of minimal genomes ERS2914955 SAMEA5130573 agalactiae_wt_SynMyco_R2_p WGA GENOMIC PCR M. agalactiae was transformed using the pMTnGm-SynMyco transposon. Two hours after the transformation, 4/5 of the culture was inoculated into 10 mL SP4 + 0.5% sodium pyruvate (Sigma-Aldrich P8574-5G) + 100 µg/mL gentamicinl. After 24 hours, the culture was collected in 500 µl of SP4. A volume of 8 µl of this culture was then inoculated into 10 mL of fresh SP4 + 0.5% sodium pyruvate (Sigma-Aldrich P8574-5G) + 100 µg/mL gentamicin. After 24 hours, the culture was collected in 500 µl of SP4 (pass 2). A volume of 8 µl of this culture was then inoculated into 0 mL of fresh SP4 + 0.5% sodium pyruvate (Sigma-Aldrich P8574-5G) + 100 µg/mL gentamicin. After 24 hours the culture was collected in 500 µl SP4. From this volume, 350 µl was taken to extract genomic DNA. The number of divisions expected was 20. The genomic DNA was isolated using the Masterpure Complete DNA & RNA Purification Kit (MC85200, Lucigen) following the protocol described by the manufacturer. 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. 250 0 F Application Read Forward 1 1 R Application Read Reverse 126 Illumina HiSeq 2000 Experimental Factor: genotype pMTnGm-SynMyco-Tn4001