title: GSM4969829: NESTED_E1; Staphylococcus aureus; Tn-Seq
library_construction_protocol: 1 µg of sheared gDNA was subjected to the NEBNext® UltraTM II DNA Library Prep Kit for Illumina® (New England Biolabs, Ipswich, MA) protocol with key modifications to the PCR amplification step. Specifically, in the PCR15 and PCR25 samples the olj511 forward primer specific to the transposon inverted repeat (IR) (PMID: 29554137) and the NEBNext Indexed reverse primers obtained from NEBNext® Multiplex Oligos for Illumina® were used to amplify the transposon-genomic junctions and add the p5 and p7 illumina adaptors as well as a unique barcode to each sample by implementing either 15 or 25 PCR cycles respectively. The cycling conditions for this PCR were 98°C for 30 sec, 98°C for 10 sec followed by 65°C for 75 sec for 15 or 25 cycles, and 65°C for 5 min. In the NESTED method, the junctions were amplified via a nested PCR consisting of two reactions, similarly to previous reports (PMID: 29554137). Here, the olj510 forward primer (PMID: 29554137) specific to the transposon but upstream to the inverted repeat region and the NEBNext Indexed reverse primers obtained from NEBNext® Multiplex Oligos for Illumina® were used in the first PCR reaction. The cycling conditions for this PCR were 98°C for 30 sec, 98°C for 10 sec followed by 65°C for 75 sec for 25 cycles, and 65°C for 5 min. In the second PCR reaction, the forward olj511 inverted region-specific primer and the same reverse primers used in the previous reaction were used to enhance the specificity towards the transposon and add the p7 illumina adaptor via 15 PCR cycles. The cycling conditions for this reaction were 98°C for 30 sec, 98°C for 10 sec followed by 65°C for 75 sec for 15 cycles, and 65°C for 5 min. The quality of the libraries was then assessed via Agilent TapeStation (Santa Clara, CA) and quantitative real-time PCR (qRT-PCR) using the olj512 primer (PMID: 29554137) specific to the transposon terminal inverted repeat. The validated libraries were then sequenced with the custom sequencing primer olj512 (PMID: 29554137) using an Illumina HiSeq 2500, Single-End 100, at the TUFTS University Core Facility. Each flow cell was loaded with six total multiplexed libraries. Thus, six samples prepared by each of the PCR methods (PCR15, PCR25 and NESTED) were loaded onto a separate flow cell. genomic DNA was extracted using the NucleoBond HMW DNA kit Detailed in 'SAMPLES'