Quantifluor dsdna system staining kit

Cosmid extraction was conducted as previously described (Gazanion et al., 2016 (link)). Briefly, total DNA was extracted from promastigotes differentiated from selected amastigotes by SDS/NaOH lysis and phenol/CHCl₃ extraction followed by EtOH precipitation. Purified total DNA was treated with RiboShredder RNase Blend (Epicentre) to remove potential RNA contaminations. Genomic DNA was removed by digesting it with Plasmid-Safe ATP-Dependent DNase (Epicentre) following the manufacturer's instructions. In addition, kinetoplastid DNA was removed by electrophoresis of DNase-treated cosmid extracts on 1% low-melting point agarose (Invitrogen) followed by excision and purification of the bands corresponding to high-molecular weight cosmid DNA (∼50 kb). Purified cosmid DNA was quantified with the QuantiFluor^® dsDNA System staining kit (Promega). Fifty nanograms of purified cosmid DNA were used for paired-end library preparation using Nextera™ DNA Sample preparation kit (Illumina) according to the manufacturer's instructions. The size distribution of Nextera libraries was validated using an Agilent 2100 Bioanalyzer and High Sensitivity DNA chips (Agilent Technologies). Sequencing libraries were quantified with the QuantiFluor^® dsDNA System and sequenced using an Illumina HiSeq system at a final concentration of 8 pM.