GSM8452962: Escherichia coli DH5a, TCMV0120 pDM2.1 pBAD33 replicate 1; Escherichia coli; OTHER
Cultures for deep-sequencing prepared as for xylE reporter gene assays with 5 mL of culture resuspended in 300 µL Puregene cell lysis solution (Qiagen, UK) before being incubated at 50°C for 10 min. 2 µL RNase A (20 mg/mL) was added and the samples were mixed by inversion 25 times before incubation at 37°C for 60 min. The samples were cooled to room temperature and 100 µL Puregene protein precipitation solution (Qiagen, UK) was added. Samples were briefly vortex, incubated on ice for 10 min and protein precipitate removed via centrifugation. The supernatant was removed to a fresh microcentrifuge tube containing 600 µL isopropanol and mixed via inversion 50 times. DNA was pelleted via centrifugation, supernatant removed and pellet washed with 600 µL 70% ethanol before a final centrifugation. The supernatant was carefully removed and the DNA resuspended in 100 µL distilled water Illumina sequencing libraries were prepared using the tagmentation-based and PCR-based Illumina DNA Prep kit and custom IDT 10bp unique dual indices (UDI) with a target insert size of 280 bp. Not additional DNA fragmentation or size selection steps were performed.