GSM3772941: untreated; Escherichia coli UTI89; RNA-Seq
The suspension was centrifuged (5 min, 4 °C) and the bacterial pellet was resuspended in 35 µL lysis buffer (lysozyme in TE buffer, 85 mg/mL). After 10 min incubation at RT and gentle vortexing every 10 sec, 1 mL TRizol ® (Thermo Fisher Scientific) was added and the isolation process was continued according to the manufacturer's protocol. 10 µg of isolated total RNA were used for DNA digestion, using the Turbo DNase (Turbo-DNA-free™ Kit, Thermo Fisher Scientific) according to the manufacturer's instructions. The RNA samples were first fragmented using ultrasound (4 pulses of 30 s each at 4°C). Then, an oligonucleotide adapter was ligated to the 3' end of the RNA molecules. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3' adapter as primer. The first-strand cDNA was purified and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified (11 PCR cycles) to about 10-20 ng/µl using a high fidelity DNA polymerase. The TruSeq barcode sequences AGGATAGG or TCAGAGCC, which are part of the 5' TruSeq sequencing adapter were used. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics).