GSM8079102: MG1655 0h -3; Escherichia coli; RNA-Seq
RNA was isolated according to the hot acid-penol method. DNA was removed with TURBO DNA-freeTM kit (Invitrogen) according to "rigorous treatment" instructions. The final clean-up was performed using phenol/chloroform/isoamyl alcohol (25:24:1) mixed with the sample in a 1:1 ratio, followed by chloroform treatment and precipitation. For cDNA synthesis, all RNA samples were first fragmented using ultrasound (4 pulses of 30 seconds, 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 to about 10-20 ng/µl using a high-fidelity DNA polymerase for 12 cycles. The TruSeq barcode sequences, which are part of the 5' and 3' TruSeq sequencing adapters, were used. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and analyzed by capillary electrophoresis.