title: GSM8688246: Sente, lib553, OD1_30C, MocklowdNTPs, SSIII, rep1; Salmonella enterica; RNA-Seq
library_name: GSM8688246
library_construction_protocol: 5 ml cells (at OD 2) were mixed with 0.2 volume stop solution (5% (v/v) phenol for RNA extraction, pH 4.5 (Roth), 95% (v/v) ethanol) and flash frozen in liquid nitrogen. Samples were thawed on ice, and cells pelleted at 4,000 g for 20 min at 4 °C. The supernatant was carefully removed, and the cells were resuspended in 0.8 ml Tris/EDTA (pH 8.0) with 0.5 mg/ml lysozyme (Roth) and incubated on ice for 10 min. 1% SDS was added to the sample. One volume phenol for RNA extraction (pH 4.5, Roth) was added to the samples for organic phase extraction, vortexted rigorously, and heated up to 64 °C for 7 min with shaking. Samples were cooled down to room temperature, the phases were separated by centrifugation at 13,000 g for 10 min and the upper aqueous fraction was collected in a new tube. One volume chloroform was added and samples were shaked for 5 min, the phases were separated by centrifugation at 13,000 g for 10 min and the upper aqueous fraction was collected in a new tube. The extraction with chloroform was repeated once more for Pseudomonas. The RNA in the sample was precipitated by addition of three volumes ethanol and 0.03 volume 3 M NaOAc pH 6.5, and storage over-night at -20 °C. The precipitated RNA was pelleted at 13,000 g for 10 min, the supernatant was removed, and the RNA was dried Remaining primers left in the reaction were digested by addition of 3 μL ExoSAP-IT (Applied Biosystems, 78205) to the 20 μL reverse transcription reaction, and incubation at 37°C for 12 min. RNA was degraded by adding 2.5 μL of NaOH 1M and 1 μL of 0.5M EDTA, and incubation at 70°C for 12 min. The base was neutralized with the addition of 1M HCl, cDNA was cleaned using MyOne silane beads. An adapter was added to the 3' end of the cDNA by ligating 50 pmoles 5iLL-22 DNA adapter using T4 RNA Ligase 1, High concentration (NEB, M0437M), DMSO 4%, ATP 1mM and PEG8000 25% at 23°C for 3 h. After clean-up with MyOne silane beads, we amplified the cDNA library for 12 cycles using Illumina Library PCR primers F and R and KAPA HiFi HotStart ReadyMix (Roche, KK2601). Amplified libraries were cleaned with Kapa pure beads (Roche, KK8002), quantified using Qubit (Life Technologies) and the distribution of library size was determined using TapeStation (Agilent Technologies).