Plasmids were isolated from S. aureus pellets with a modified protocol of QIAprep Spin Miniprep Kit (Qiagen Cat. #27104), as previously described (Aviram et al., 2022 (link)). 250 ng of each sample were used as input for PCR, using the Phusion DNA Polymerase (Thermo Cat. #F530L) with primers NA162/NA163 for the spacer-library experiments, and with NA101/NA102 or NA169/170 for type III-A or type II-A naïve spacer acquisition, respectively (see Table S7 for a full list of primers used in this study).
Spacer library PCRs were analyzed by agarose gel electrophoresis, bands were excised and purified using QIAquick gel extraction kit (Qiagen Cat. #28704). Naïve spacer acquisition PCRs underwent cleanup with the MinElute PCR Purification Kit (Qiagen Cat. #28004) and size selection using PippnHT 3% cassette with a timed protocol set at extraction between 26 and 35 min. Size selected products were then prepared for sequencing with the TrueSeq Nano DNA Library Prep protocol (Illumina). For maintaining the small sized product, 2.2× Sample Purification Beads (Ilumina) were used after end repair. Illumina libraries underwent high-throughput sequencing with the MiSeq platform.
Spacer library PCRs were analyzed by agarose gel electrophoresis, bands were excised and purified using QIAquick gel extraction kit (Qiagen Cat. #28704). Naïve spacer acquisition PCRs underwent cleanup with the MinElute PCR Purification Kit (Qiagen Cat. #28004) and size selection using PippnHT 3% cassette with a timed protocol set at extraction between 26 and 35 min. Size selected products were then prepared for sequencing with the TrueSeq Nano DNA Library Prep protocol (Illumina). For maintaining the small sized product, 2.2× Sample Purification Beads (Ilumina) were used after end repair. Illumina libraries underwent high-throughput sequencing with the MiSeq platform.
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