GSM7928258: WT replicate 2; Bacillus subtilis subsp. subtilis str. 168; Tn-Seq
Cell lysis, ammonium acetate precipitation of proteins, followed by isopropanol precipitation of gDNA 6 µg gDNA was digested with 3 units of MmeI for 2.5 hours at 37 °C, then treated with 6 units of antarctic phosphatase for 1 hour at 37 °C. DNA was then extracted with 300 µl  phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated with 50 µL 3M sodium acetate and 500 µL isopropyl alcohol. Dried pellets were dissolved in 27.5 µL 2 mM Tris-HCl pH 8.5. The DNA was ligated with one volume of 200-µM annealed adapters HRH-Tn1 and HRH-Tn2 in 1 mM tris-Cl pH 8.5. The ligated DNA was purified with the Qiagen PCR purification kit and amplified with the primers HRH-Tn3 and HRH-Tn4.