native Tn-seq analysis of ISAba13 in Acinetobacter baumannii AB5075
native Tn-seq analysis of ISAba13 in Acinetobacter baumannii AB5075
A. baumannii AB5075 is a clinical strain isolated from an infected tibia.Jacobs, A. C. et al. AB5075, a Highly Virulent Isolate of Acinetobacter baumannii, as a Model Strain for the Evaluation of Pathogenesis and Antimicrobial Treatments. (2014) doi:10.1128/mBio.01076-14. Cells were grown in liquid LB medium at 37 degrees with shaking. Two biological replicates were done for each genetic background. Following overnight growth, cells were sub-cultured in 50 ml LB and grown to mid exponential phase (OD650 = 0.5). Genomic DNA was extracted from 13 ml of the culture using a DNeasy Blood & Tissue kit (Qiagen) according to the  manufacturer's instructions for Gram-negative bacteria. The optional RNase A step was included and the final DNA elution was done using 100 µl of dH2O. The extracted DNA was further purified with Agencourt AMPure XP magnetic beads, using a 1.8:1 ratio of beads:DNA, according to the manufacturer's instructions. After washing DNA bound beads twice, with 200 µl of 70% (v/v) ethanol, DNA was resuspended in dH20.  Our procedure was derived from method of Long et al. NEBnext dsDNA fragmentase was used to digest 6 µg of genomic DNA. Reactions were incubated for 10-15 minutes at 37 °C and stopped with 5 µl 0.5M EDTA. Fragmented DNA, between 400 - 650 bp in size, was extracted from a 1% (w/v) agarose gel following electrophoresis. Gel slices were stored at -20 °C for >30 minutes and DNA extracted using the Qiagen minelute kit according to manufacturer's instructions with an extra PE buffer wash. Next, 1 µg of DNA was mixed with 7 µl NEBnext Ultra II end prep reaction buffer (NEB) and 3 µl Ultra II end prep enzyme mix (NEB), and incubated at 20 °C for 30 min, followed by 65 °C for 30 min. DNA was purified using Agencourt AMPure XP magnetic beads at a 1:1 ratio of beads:DNA. Next, 48 µl of 100 µM stock DNA adaptors 1.2 and 2.2 (see associated publication for details) were mixed with 4 µl 50 mM MgCl2, at 95 °C for 10 min, and allowed to cool to room temperature overnight. Adaptor 1.2 has a 3' C3-amino modification to protect against exonuclease degradation and can be ligated to DNA 5' ends. The adaptor 2.2 can be ligated to DNA 3' ends. Adapters were attached to DNA fragments using either a Fastlink DNA ligation kit (Cambio) or, following discontinuation of this reagent, the NEBnext ultra II ligation module (NEB). After ligation, products were purified with a 0.7:1 ratio of AMPure XP beads to DNA. Purified products were used as template for 50 µl PCR reactions containing 1x GoTaq (Promega), 0.3 µM each of oligonucleotides JelAP1, and ether ISAba13_out or insH3_out, and 5 % (w/v) DMSO. For each experiment, 8 reactions were done using 100 ng of DNA template in each. The PCR conditions comprised an initial 95 °C denaturation step, followed by 20 cycles of 95 °C (30 s), 58 °C (30 s) and 72 °C (45 s), and a final 72 °C step for 5 minutes. The JelAP1 oligonucleotide anneals to adaptor 2.1 and results in extension products directed towards the isolated genomic DNA fragment. The ISAba13_out and insH3_out oligonucleotides anneal close to the 3' end of their cognate insertion sequence. When used with JelAP1, they result in extension products across the insertion boundary with chromosomal DNA, towards the site of adaptor 2.1 attachment. Thus, all products of the PCR reaction capture boundaries between ISAba13 and chromosomal DNA at insertion sites. Following PCR, each set of 8 equivalent reactions was pooled and products separated on a 2 % (w/v) agarose gel. Products in the region of 400-650 bp were excised in a gel slice that was then stored at -20 °C for 30 minutes. The DNA was then recovered using a minelute gel extraction kit (Qiagen) using 4 PE buffer washes.   In a second round of PCR reactions we used an oligonucleotide (one of the “Ap_P7_tagged” variants) that anneals to the JelAP1 sequence and introduces a DNA barcode. This was in conjunction with a mixture of the 4 “staggered” oligonucleotides, all annealing to the same site towards the 5' end of the ISAba13 sequence, and containing different stager lengths to improve complexity of the final library. Resulting PCR products should contain the final 78+ bp (depending on the stagger length and insertion element) of the insertion sequence 5' end. The PCR conditions were the same as those used in step one above, except that the cycle number was reduced to 8. After PCR, DNA was recovered using a1:0.75 ratio of AMPure XP beads. Long, J. E. et al. Identifying essential genes in mycobacterium tuberculosis by global phenotypic profiling. in Methods in Molecular Biology vol. 1279 79–95 (Humana Press Inc., 2015).