GSM2579559: treated with Roxithromycin 2ug/mL; Mycolicibacterium smegmatis; Tn-Seq
The Nextera DNA sample preparation kit (Illumina) was used to fragment isolated genomic DNA, with minor modification; 10 ng/mL DNA was used as input for larger fragment generation, and the tagmentation reaction was purified with a Qiagen PCR purification kit with a 57 mL elution. Three 25 mL real-time-qPCR reactions were prepared per primer pair per condition with 9 mL of the eluate. Quanta's PerfeCTa® SYBR® Green SuperMix with a final concentration of 0.8 mM per primer was used for the reactions. A primer targeting the P7 tagmentation (P7tag) was paired with primers targeting either end of the FMycoMarT7 transposon (Accession: AF411123); NX_pMMT7_out1 and NX_pMMT7_out2. Reactions were run in a CFX96 RT-PCR machine (Bio-Rad) with decreasing annealing temperatures: 94 ºC for 30 s, Annealing for 30 s (60 ºC for 5 cycles, 58 ºC for 5 cycles, and then 55 ºC for 35 cycles), 72 ºC for 30 s.  The live readout was monitored, and the PCR was stopped when 6 cycles had passed since the signal increased above background (~23 cycles total). All reactions were then separated on a 2% agarose-TAE gel with the smear between 250-500 bp cut out, the triplicates combined, and the DNA purified with the Qiagen Gel purification kit with a 45 µL elution. Eluates were quantified with the Qubit dsDNA HS assay kit, and the products from the two reactions pooled at equal amounts to a final concentration of ~1.4 ng/µL. Note, if there wasn't enough DNA from both reactions for an equal pool, the more concentrated partner was added at higher amounts to get the desired final concentration. The Nextera sample preparation kit protocol was then continued. A second PCR was set up with 24 µL of pooled DNA as suggested, but 1 µL of a custom P5 primer (phiP5) at 20 µM was used and the number of cycles increased to 10. The Nextera indexing kit P7 primers were used to barcode the individual conditions. Samples were combined and sequenced using the MiSeq reagent kit v2 (Illumina) with 50 bp reads using the custom P5 primer phiMMT7seq_1. Around 3 million reads were obtained per condition.  More details on the extraction and preparation protocols are available on the series record in the file "Protocol.pdf".
GEO Accession
GSM2579559