GSM9422058: ShootTnA; Burkholderia contaminans; Tn-Seq
Plant material was removed from the Falcon tube, the bacterial culture was pelleted by centrifugation at 3000 rpm for 5 min, and media decanted. Three cell pellets were obtained for scenarios a, b roots, b shoots, c roots, c shoots, and timepoint 0 for a total of 18 cell pellets for DNA extraction. 19 Pellets of DNA were extracted using the DNeasy Powerlyzer PowerSoil kit Sequencing library preparation of samples for the Tn-Seq experiment followed the protocol used in Ganesan et al. 2021 with modifications. The NEBNext® Ultra™ II DNA Library Prep Kit for Illumina was used but at half reaction volumes. Specifically, 400 ng of input DNA in 13 mL was processed for each of the 19 samples. To the DNA for each sample, 3.5 mL NEBNext Ultra II FS Reaction Buffer and 1 mL of NEBNext Ultra II FS Enzyme Mix was added. This mixture was incubated for 15 minutes at 37°C and then 30 minutes at 65°C to fragment the DNA and terminate the fragmentase activity. Next, 15 mL of NEBNext Ultra II Ligation Master Mix, 0.5 mL of NEBNext Ligation Enhancer, and 1.25 mL of NEBNext Adaptor for Illumina was added to this mixture and incubated for 15 minutes at 20°C. 1.5 mL USER® Enzyme was added to this mixture and incubated at 37°C for 15 minutes with heated lid set to ≥ 47°C. This 37.75 ml reaction volume was brought to 100 ml using 0.1X TE and size selected with SPRI Ampure magnetic beads with right side selection of 0.3X and a left side selection of 0.15X. The size selected fragments concentrated into 7.5 mL were PCR amplified using 2.5 mL of a forward primer specific for the transposon that also harbored a 5' biotin label, 2.5 mL of a P7 barcoded sequencing primer NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1 and 2), and 12.5 mL of NEBNext Ultra II Q5 Master Mix. The thermocycler denature, anneal and extension times were an initial 98°C for 30 seconds, then ten rounds of 98°C for 10 seconds, 65°C for 30 seconds, and 72°C for 30 seconds, with a final extension of 72°C for 2 minutes. This reaction was cleaned up by 0.9X left side selection with SPRI Ampure magnetic beads. 12.5 mL of the biotin tagged product was hybridized to streptavidin magnetic beads to select for reads harboring the transposon primer. The beads were washed with 1X binding and wash buffer to remove any products harboring the biotinylated transposon-specific DNA fragments. A second PCR was carried out using the hybridized biotinylated transposon-streptavidin beads in 7.5 mL of 0.1X TE, 2.5 mL of a forward primer that included the P5 adapter sequence followed by a random hexamer sequence and the transposon specific primer sequence, 2.5 mL of the corresponding P7 barcoded sequencing primer NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1 and 2 ) used in the first PCR reaction for the respective sample, and 12.5 mL of NEBNext Ultra II Q5 Master Mix. Each library was quantified using the Qubit DNA High Sensitivity (HS) kit and the Agilent Bioanalyzer DNA HS kit. The 19 barcode sequencing reads were pooled to achieve a 20 nM sample in 40 mL and shipped to Azenta GeneWiz for sequencing on the Illumina Nextseq platform.