title: NextSeq 500 paired end sequencing; DNASeq of different Mycoplasma pneumoniae isolated clones presenting a genomic deletion produced by a multi-recombinase engineering protocol
library_construction_protocol: Cell suspensions (70 µl) from M129-GP35-PtetCre or M129-GP35 electrocompetent cells were mixed with 0.5 nmol of the editing primer selected (i.e., 5 µl of a 100 µM oligo solution) and 2 µg of the desired selector vector,. In all cases, a control transformation only with the selector vector was carried out to estimate the frequency of spontaneous plasmid insertion in the absence of editing oligo. After the electroporation pulse, cells were harvested from the cuvette in Hayflick medium already supplemented with aTc and allowed to recover at 37ºC for 2 h. The entire transformation volume was then inoculated in a 75-cm2 flask containing 25-ml Hayflick supplemented with puromycin and aTc, to induce expression of SSR-A, which mediates plasmid integration. After 24 h, cells were scraped from the flasks and seeded onto puromycin-selective Hayflick-agar plates. Colonies were picked from puromycin-selective Hayflick-agar plates and inoculated in 96 well plates filled with 200 µl of puromycin-supplemented Hayflick medium per well. After PCR screening of positive clones carrying the intended modification, plasmid backbone was excised from the edited area (except in the case of M129-GP35-PtetCre Δ30kbNE region::pLoxPuro clone). To this end, cells were grown in tissue culture flasks to prepare electrocompetent cells and the resulting cell suspensions (70 µl of each) were mixed with 2 µg of a suicide vector termed pGentaVcre. A control transformation with no plasmid was always performed in parallel. After the electroporation pulse, cells were allowed to recover at 37ºC for 2 h before inoculating one-fifth of the transformation in a 75-cm2 flask containing 25-ml Hayflick supplemented with gentamicin. Flasks were incubated at 37ºC for 5 days, a timeframe long enough to kill non-transformed cells and to excise the selector plasmid from the edited area in cells that received the pGentaVcre suicide vector. After this incubation, survivor cells were scraped from the flask and seeded onto non-selective Hayflick-agar plates. Colonies grown on 96-well plates containing 200 µl of non-selective Hayflick were picked and screened by PCR to the excision of the selector plasmid from the edited area.  Clones carrying the intended modifications confirmed by PCR were grown in T25 flasks in Hayflick medium until late exponential phase and genomic DNA was extracted using MasterPure DNA purification kit (Lucigen) following manufacturer's instructions. Genomic DNA was quantified using Qubit® dsDNA BR (Invitrogen, Life Technologies) and its integrity was assayed with an agarose gel. gDNA fragmentation was done using the Covaris S2 instrument (Covaris Inc.) adjusting the settings as follows: 10% duty cycle, intensity 5, and 200 cycles per burst for 40 seconds (for 600bp size distribution). Libraries were prepared using the NEBNext® Ultra DNA Library Prep for Illumina® kit (ref. E7370) according to the manufacturer's protocol. Briefly, 100 nanograms of DNA were subjected to end repair, addition of “A” bases to 3' ends, ligation of adapters and USER excision. All purification steps were performed using AgenCourt AMPure XP beads (ref. A63882, Beckman Coulter). Library amplification was performed by PCR using NEBNext® Multiplex Oligos for Illumina (Index Primers Set 1, ref. E7335), (Index Primers Set 2, ref. E7500), (Index Primers Set 3, ref. E7710) or/and (Index Primers Set 4, ref. E7730). Final libraries were analyzed using Agilent DNA 1000 chip to estimate the quantity and check size distribution, and were then quantified by qPCR using the KAPA Library  Quantification Kit (ref. KK4835, KapaBiosystems) prior to amplification with Illumina's cBot.