Ribo zero magnetic kit for bacteria

Total RNA was extracted using AllPrep DNA/RNA Mini Kit (Qiagen, Germany) according to the manufacturer's instruction. Lysis buffer RLT plus was supplemented with 2% (v/v) 2 M Dithiothreitol (DTT) (Sangon Biotech, China). Lysis buffer RLT plus (600 μl) was added to each cell pellet. Cell pellets of PM and PMC were homogenized for 120 s (5.0 m/s, 3 × 40 s with 2 min intervals on ice) in a FastPrep-24 (MP Biomedicals, USA) with 0.5 g RNase free beating beads (0.1 mm, Sigma, USA). Cell pellets of MM were homogenized at 5.0 m/s for 20 s without beating beads. Residue DNA digestion, verification of absence of DNA, and RNA purification were carried out as previously described (Liu et al., 2014 (link)). RNA was finally dissolved in 75 μl RNase free H₂O. RNA integrity values (RIN) were determined by an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) according to the manufacturer's instruction (Figure S2). Aliquots of total RNA were kept for quantitative real-time PCR (qRT-PCR), and the remainder (≥10 μg) was sent to Beijing Genomics Institute (BGI) (http://www.genomics.cn, China) on dry ice for mRNA enrichment by using the Ribo-Zero™ Magnetic Kit for Bacteria (Epicenter, USA).

M. extorquens wild-type strain DM4 was grown aerobically at 30 °C in M3 medium supplemented with methanol or DCM provided at 10 mM as the sole source of carbon and energy, in independent duplicates of 220 mL in 1.2 L Erlenmeyer flasks closed with gas-tight screw caps with Supelco Mininert R valves (Thermo Fisher Scientific, Illkirch, France), as described previously [31 (link)]. Cells were harvested at mid-phase (OD at 600 nm of 0.15), and RNA was extracted and treated with a TURBO DNA-free kit (Thermo Fisher Scientific, Illkirch, France) to remove residual genomic DNA. Ribosomal RNAs were depleted using the Ribo-Zero™ Magnetic Kit for Bacteria (Epicentre, Madison, WI, USA). Integrity of RNAs was validated after high-resolution automated electrophoresis of the RNA samples using the Agilent 2100 Bioanalyzer system. Two biological replicates were performed for each of the two tested conditions.

Total RNA from bacterial cultures was extracted using the SV Total RNA Isolation System kit (Promega) following the manufacturer's instructions for Gram-negative Bacteria. Infected plant RNA extractions were carried out as described (Cruz et al., 2014 (link)). RNA concentration and quality was measured using the Agilent 2100 Bioanalyzer. For rRNA depletion, 2.5 μg of RNA were treated with the Ribo-zero^(™) magnetic kit for bacteria (Epicenter). Three biological replicates per condition were subjected to sequencing on an Illumina-Solexa Genome Analyzer II apparatus in the Shanghai PSC Genomics facility using multiplexing and kits specially adapted to obtain 100 bp paired-end reads in stranded libraries. Raw sequencing data is available in the Sequence Read Archive under the accession code SRP096020.

Culture samples were harvested directly into double the volume of RNAprotect Bacteria Reagent (QIAGEN), incubated for 5 minutes, and then centrifuged. Cell pellets were stored at −20 °C before RNA extraction on the following day. Total RNA was isolated using RNeasy Mini columns (QIAGEN) with a modification to the manufacturer’s instructions. Briefly, cells were lysed in the presence of 300 mg acid-washed glass beads (150–600 μm diameter, Sigma) using a Fast Prep Lyser (Eppendorf) with three cycles of 1 min at maximum speed and one cycle of 7 min, cooling the samples on ice for 2 min before and between cycles. On-column DNA digestion was performed using the RNase-Free DNase Set kit (QIAGEN). RNA sample integrity was verified using a NanoDrop^TM, (absorbance ratios 260/280 nm and 260/230 nm ≥1.8) and a Bioanalyzer 2100 (RNA Integrity Number (RIN) ≥7). Samples of total RNA (≥5 μg, measured on a Qubit) were submitted to a commercial provider (Beijing Genomics Institute) for transcriptome sequencing. Prior to sequencing, all samples were treated with DNaseI (New England Biolabs® Inc) and then enriched for mRNA molecules via ribosome-depletion using the Ribo-Zero™ magnetic Kit for bacteria (Epicentre). Sequencing was performed using an Illumina HiSeq 2000 with TruSeq V3 sequencing kits.