Microb express bacterial mrna purification kit

Ribosomal RNA was depleted using a MICROB Express Bacterial mRNA Purification kit (Ambion), following the manufacturer’s protocol. Integrity and quality of the ribosomal depleted RNA was checked with Agilent Bioanalyzer 2100 (Agilent Technologies). RNA sequencing was carried out by Sistemas Genómicos (https://www.sistemasgenomicos.com/web_sg/) with the Next Generation Sequence (NGS) platform Illumina using the Illumina HiSeq 2000 sequencing instrument (Illumina). Ribosomal-depleted samples were used to generate whole transcriptome libraries following the manufacturer's recommendations for sequencing on this NGS platform. Amplified cDNA quality was analyzed by the Bioanalyzer 2100 DNA 1000 kit (Agilent Technologies) and quantified using the Qubit 2.0 Fluorometer (Invitrogen).

Removal of 16S and 23S rRNA from total RNA was performed using MicrobExpress™ Bacterial mRNA Purification Kit (Ambion) according to the manufacturer’s protocol with the exception that no more than 5 μg total RNA was treated per enrichment reaction. Each RNA sample was divided into multiple aliquots of ≤5 μg RNA and separate enrichment reactions were performed for each sample. Enriched mRNA samples were pooled and run on the 2100 Bioanalzyer (Agilent) to confirm reduction of 16S and 23S rRNA prior to preparation of cDNA fragment libraries.

Total RNA samples were extracted from the cultures with the use of the RiboPure Bacteria kit (Ambion, Lithuania) as instructed by the developer’s guidelines. Afterward, the quantity and quality of the extracted RNA were verified with the use of the NanoDrop 2000c spectrophotometer (Thermo Scientific, USA) at optical wavelengths of 260 and 280 nm. Purification of the total RNA from 16S and 23S rRNA was carried out using the MICROBExpress bacterial mRNA purification kit (Ambion, Lithuania) following the developer’s recommendations. Thereafter, the effectiveness of sample purification was determined on the Bioanalyzer 2100 (Agilent, Germany) with the RNA 6000 Nano LabChip kit (Agilent Technologies, Lithuania).
The library preparation from the extracted RNA samples included an enzymatic fragmentation step with the use of the Ion Total RNA Seq kit V2. Thereafter, the Ion Xpress RNA-Seq Barcode 01-16 kit was used for RNA fragment barcoding. cDNA sequencing was then performed using the Ion 318 Chip kit V2 on the Ion Torrent PGM sequencer (Life Technologies, USA).

Total RNA was extracted using TRIzolTM (Thermo Fisher Scientific, Waltham, MA, USA) protocol (122 (link)). Four independent RNA extractions were obtained for each strain for each time point. The concentration of RNA samples was determined using QubitRNA HS Assay kit (Invitrogen). Ribosomal RNA was depleted using the MICROB Express Bacterial mRNA purification kit (Ambion, Foster City, CA, USA). The integrity and quality of the RNA were checked by an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).

Total RNA extraction of EC-12 was performed as described above. The total RNA obtained was separated using electrophoresis through 1.5% agarose gels (1× Tris-borate-EDTA buffer) and 23S rRNA and 16S rRNA were excised using a sterilized blade. Zymoclean Gel RNA Recovery Kit (Zymo Research, Orange, CA, USA) was used for the recovery/purification of 23S rRNA and 16S rRNA from the gel, according to the manufacturer’s instructions, with the following modification: Mini Plus Column (Viogene, Taipei, Taiwan) was used instead of the supplied Zymo-Spin IC Column to yield a higher RNA concentration. Fragmentation of RNA, purification of fragmented RNA, and assessment of size were performed as described above.
Purified mRNA was obtained from total RNA by sequential removal of rRNA and small RNA with the MICROBExpress Bacterial mRNA Purification Kit (Ambion, Tokyo, Japan) and the MEGAclear Kit (Ambion), according to the manufacturer’s instructions.
All RNA molecules were eluted in nuclease-free water.

Overnight cultures in M9 glucose were inoculated in 100 mL fresh M9 glucose to a final OD₆₀₀ of 0.02 and incubated at 37° with shaking. Two biological replicates were performed for each strain. Cells were collected by centrifugation at the early exponential (OD₆₀₀ ∼0.3), mid-exponential (OD₆₀₀ ∼0.8), transition to stationary (OD₆₀₀ ∼1.6), stationary (16 hr, OD₆₀₀ ∼2), and late stationary (48 hr, OD₆₀₀ ∼1.6) phases of growth. RNA was extracted using TRIzol (Invitrogen), following the manufacturer’s protocol. Total RNA was treated with DNase I (Invitrogen, 18068-015) according to the manufacturer’s protocol. Further precipitation of RNA and ribosomal RNA cleanup was achieved using the MICROBExpress bacterial mRNA purification Kit (Ambion, AM1905) according to the manufacturer’s protocol. RNA concentration was determined using a Nanodrop 2000 (Thermo Scientific) and quality was checked by visualization on agarose gels.