GSM3326154: FUSE_37B: FUSE of cells treated with DMS at 37°C (replicate B); Listeria monocytogenes EGD-e; OTHER
10 ml of bacterial culture was mixed with 5 ml of 1:20 phenol:ethanol solution and centrifuged at 7000 g for 10 minutes. The pellet was resuspended in the Disruption solution (10% glucose, 12.5 mM Tris-HCl pH 7.6, and 5 mM EDTA) and immediately transferred to 2 ml screw-capped tubes with roughly 0.4 g glass beads and 500 μl of acid phenol (pH 4.5). The bacteria were disrupted using a mini bead beater (Biospec products) for 30 s. After centrifugation (5 min, 12000 g) RNA was recovered by addition of 1 ml of TRI Reagen Solution (Thermo Fisher) and 100 μl of chloroform, followed by centrifugation. Samples were thereafter subjected to two additional chloroform extractions. The aqueous phase was precipitated by adding isopropanol (0.7×) and incubation at −20°C for 20 min. For collection of the pellet, the RNA samples were centrifuged for 25 min. The pellet was washed with 80% ethanol and dissolved in 50 μl of RNase-free water. After modification with DMS, RNA was treated with DNase I (Roche) and purified on RNeasy MinElute columns (Qiagen). RNA was treated with RNA 5'-Polyphosphatase (Epicentre) and once again purified on the columns. RNA was depleted of ribosomal fraction with Ribo-Zero rRNA Removal Kit (Illumina), and purified on RNeasy columns. RNA solution was concentrated on SpeedVac Concentrator (ThermoFisher Scientific) to 4.5 µl, mixed with 1 µl of 15 µM PAGE-purified 5'RA ribooligonucleotid (UCCCUACACGACGCUCUUCCGAUCU), heated for 3 min at 65°C and cooled on ice. The ligation reaction was assembled with T4 RNA Ligase 1 (NEB) and 10% DMSO, and proceeded for 2 hours at room temperature. RNA was ethanol precipitated, dissolved in 9 µl of mQ water and fragmented with RNA Fragmentation Reagents (Thermo Fisher Scientific) for 3.5 min at 70°C. Following the fragmentation, RNA was run on 6% denaturing polyacrylamide gel, and fragments ranging in size from 125 to 400 nucleotides were isolated from the gel. RNA fragments were dephosphorylated with T4 Polynucleotide Kinase (Thermo Fisher Scientific) in the dephosphorylation buffer (100 mM MES pH6.0, 10 mM MgCl2) and purified on RNeasy columns according to the modified protocol (100 µL of RNA solution was mixed with 350 µL RLT buffer and 550 µL 96% ethanol). The modified protocol preserves fragments shorter than 200 nucleotides and is used at the steps following RNA fragmentation. 3'DA adapter (5phos/AGATCGGAAGAGCACACGTCTGAACTCCAG/3ddC) was adenylated with 5' DNA Adenylation Kit (NEB). RNA solution was concentrated to 4.5 µL and mixed with 1 µl of 15 µM adenyl-3'DA. Ligation reaction with T4 RNA Ligase 2, truncated (NEB) and 25% PEG8000 was allowed to proceed for 2 hours at room temperature. RNA was ethanol precipitated, dissolved in 20 µL of mQ water and treated with 1.6x vol:vol AMPure XP beads (Beckman Coulter) to remove not ligated adaptors. cDNA was synthesized with TGIRTIII enzyme (InGex): RNA solution was concentrated to 4.5 µl, mixed with 1 µl of 1 µM RT primer and heated for 2 min at 80°C. The primer was annealed for 5 minutes at room temperature, and the reverse transcription reaction was assembled in RT buffer (50 mM Tris-HCl pH8.3, 75 mM KCl, 3 mM MgCl2) with 1 mM dNTPs mix, 5 mM DTT and 100U of TGIRTIII enzyme. The reverse transcription proceeded for 2 hours at 57°C. After that 1 µl of 5N NaOH was added to the reaction and the reverse transcriptase was inactivated by heating at 95°C for 3 min. The first strands of cDNA were ethanol precipitated, dissolved in mQ water and cDNA libraries were amplified by 2 rounds of PCR. The first 12 cycles of PCR were performed with primers LibAmp_F (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTC) and LibAmp_RPI_R (CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCT), which introduced sequences identical to Illumina TruSeq adapters (Oligonucleotide sequences © 2018 Illumina, Inc. All rights reserved) to cDNA. The product of the first PCR reaction was purified with 1x vol:vol AMPure XP beads and used as matrix for the second 6-8 cycles PCR reaction with primers Enrich_F (AATGATACGGCGACCACCGAGATC) and Enrich_R (CAAGCAGAAGACGGCATACGAGAT). The product of the second PCR reaction was sequentially purified with RNeasy PCR Purification Kit (Qiagen) and 1x vol:vol AMPure XP beads.
GEO Accession
GSM3326154