GSM4969835: nCATRAs_S1; Staphylococcus aureus; Tn-Seq
The previously published nCATS protocol was modified in this paper to enrich for the transposon-genomic insertions such that only one end of the region of interest was cleaved as opposed to excision of the region of interest as described in the nCATS protocol (PMID: 32042167). In our modified nCATRAs method, gDNA extracted from the TnSeq library was sheared using Covaris g-tubes to 6,000bps, which resulted in DNA fragments between 6kb to 10kb in size. Two samples each containing 6ug of fragmented gDNA were separately dephosphorylated using Quick CIP (NEB) and incubated at 37°C for 15 mins. Two crRNA probes targeting two sites on the 5' end of the transposon were used to direct Cas9 to the region. Cas9-cleaved DNA fragments were ligated to the Oxford Nanopore adaptors and the Ligation Sequencing Kit (LSK-109) protocol was followed according to the manufacturer. The two starting samples were pooled in the final elution step prior to loading onto the MinION flow cell. One MinION flow cell was used per pool of two samples. genomic DNA was extracted using the NucleoBond HMW DNA kit Detailed in 'SAMPLES'