GSM8431617: 1043, liquid culture, 4.5hrs, 37 degrees celcius, rep1; Serratia marcescens; RNA-Seq
Cultures were mixed with 10 ml ice-cold stop solution (19% ethanol, 1% acidic phenol pH 4.3) and incubated on ice for 30 min. Cells were then recovered by centrifugation, resuspended in 1 mL TRIzol and transferred into a 2 mL phase-lock tube, and 400 μL chloroform was added and immediately mixed for 10 s. After incubation for 2 min at room temperature, the suspension was separated by centrifugation for 15 min at 20,000 g. The upper phase containing the RNA (∼500 μL) was transferred into a 1.5 mL microcentrifuge tube, 450 μL isopropanol was added, and the suspension was mixed by inverting and then incubated at room temperature for 30 min. The precipitated RNA was recovered by centrifugation for 30 min at 20,000 g and washed in 350 μL 70% ethanol. Following a further centrifugation step (10 min, 20,000 g), the pellet was air-dried, resuspended in RNase-free water and stored at -80°C. ﻿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 to about 10-20 ng/µl using a high fidelity DNA polymerase.  cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and was analyzed by capillary electrophoresis.