Zymobiomics rna miniprep kit

Bacterial cells from 2 mL cultures were collected by centrifugation for 3 min after addition of ½ volume of frozen killing buffer (20 mM Tris-HCl pH 7.5, 5 mM MgCl₂, 20 mM NaN₃) to the culture sample. The cell pellets were frozen in liquid nitrogen and stored at −80°C. For experiments performed in iron-depleted conditions, RNAs were extracted from pellets harvested at an OD600 of 0.8. For experiments performed in 1% haemoglobin-enriched conditions, RNAs were extracted from pellets harvested when bacterial concentration reached 10⁹ CFU/ml. Experiments were carried out from three independent cultures per biological condition. The ZymoBIOMICS RNA Miniprep Kit (Zymo Research) was used for total RNAs extraction, then 5 µg of RNA extracts were treated using DNase I (Qiagen) to remove residual genomic DNA and purified using the ZymoBIOMICS RNA Miniprep Kit. The RNA concentration was measured using a NanoDrop 1000 Spectrophotometer (NanoDrop Technologies, Inc.) and quality was assessed by electrophoresis on denaturing formaldehyde agarose gel.

Extraction of total RNA was done using four different commercially available kits, namely the RNeasy PowerMicrobiome kit, (QIAGEN GmbH, Hilden, Germany), the Stool Total RNA purification kit (Norgen Biotek crop. Thorold, Canada), ZymoBIOMICS™ RNA Miniprep kit (Zymo Research, Irvine, CA, USA), and the NucleoSpin RNA stool kit (Macherey-Nagel GmbH, Düren, Germany). The RNA extraction was conducted in triplicate, yielding twelve samples (i.e., three per combination) per each animal type. The combinations were the following: RL stabilization + short storage (4 °C, 24 h); RL stabilization + long storage (4 °C, 24 h followed by −80 °C, 2 weeks); ZM stabilization + short storage; ZM stabilization + long storage. The tubes containing samples were thawed on ice and then vortexed for homogenization. The content of each tube was transferred into individual 2 ml Eppendorf tubes and centrifuged with 15,000 × g (Eppendorf Centrifuge 5424 R) for 5 min at room temperature (25 °C) to pellet out the feces and to remove the supernatant, which could contain traces of stabilizers. The RNA extraction from all samples was carried out following kit protocols with minor adjustments: Firstly, 150 mg of sample were used as initial starting material, and secondly, 80 μl of RNAse-free water was used instead of 100 µl for the elution of all the extracted RNA.

Bacterial RNA was extracted using the ZymoBIOMICS RNA Miniprep Kit (Zymo Research, Irvine, CA). Sequencing and library construction were performed on the Illumina Hiseq 2500 with 101 bp paired-end. Ribosomal RNA was removed using the Ribo-Zero™ rRNA Removal Kit (Bacteria) (Epicentre, Madison, WI). Libraries were prepared with the TruSeq RNA Sample Prep kit v2 (Illumina). RNA-sequenced reads were mapped on the reference genome of Bifidobacterium breve (NZ_AP012324.1) using STAR with alignIntron MAX 1 [19 (link)]. Then, the mapped reads were used to calculate read counts of genes using cufflinks [23 (link)], and the gene list was inputted into Cytoscape plug-in ClueGO v2.5.4 [22 (link)] to annotate functionally grouped networks. Functionally related GO terms for biological processes in Escherichia coli (version: November 18, 2016) were grouped based on a kappa score greater than 0.4 with a network specificity of 4–10. Statistical significance was calculated using two-sided hypergeometric tests, and the false discovery rate was corrected using the Bonferroni step down method.