title: native Tn-seq analysis of ISAba13 in Acinetobacter baumannii AB5075
design: native Tn-seq analysis of ISAba13 in Acinetobacter baumannii AB5075
library_construction_protocol: 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 °