GSM8172009: Bil_system_R1; Escherichia coli; RNA-Seq
E. coli expressing the Bil system under control of a tetracycline-inducible promoter was grown to mid-log phase with 25 ng/ml aTc as inducer. Caulobacter sp. Root343 was grown to mid-log, late-log or early stationary phase. Samples corresponding to an OD600 of 2 were taken and immediately mixed with ¼ volume of stop mix (95% ethanol, 5% saturated phenol) and frozen in liquid nitrogen. The samples were then thawed on iced followed by centrifugation at 4,000 g and 4°C for 10 min. The supernatant was discarded and the cell pellets resuspended in 1 ml of TRIzol (Thermo Scientific) followed by RNA extraction according to the manufacturer's protocol. The resulting RNA pellet was dissolved in 40 µl of water. To get rid of contaminating DNA, 0.5 µl of water, 5 µl DNase I buffer with MgCl2 (Thermo Scientific), 0.5 µl of RNase inhibitor (Thermo Scientific) and 4 µl DNase I (Thermo Scientific) was added and the mixture incubated at 37°C for 30 min. The DNase-digested RNA was subjected to acidic phenol-chloroform extraction and finally dissolved in 30 µl of water. Extracted RNA was depleted of ribosomal RNA using a commercial rRNA depletion kit for mixed bacterial samples (Lexogen, RiboCop META, #125). The depleted RNA samples were fragmented using ultrasound (2 pulses of 30 s 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 with the 3' adapter as primer. After purification, the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified using a high-fidelity DNA polymerase and the barcoded TruSeq-libraries were pooled in approximately equimolar amounts. Sequencing of pooled libraries, spiked with PhiX control library, was performed at a minimum of 10 million reads per sample in single-ended mode with 100 cycles on the NextSeq 2000 platform (Illumina).