Rna 6000 nano reagent

Total RNA was extracted from each sample using RNeasyPlus Micro Kit (Qiagen, Germany) according to manufacturer’s protocol. Purity (A₂₆₀/A₂₈₀), RNA integrity number (RIN) and concentration of extracted total RNA were assessed using Agilent 2100 Bioanalyzer with RNA 6000 Nano Reagents (Agilent Technologies, Santa Clara, USA). Only samples with RIN 7.0 or higher were selected for the gene expression experiment. 100 ng of total RNA of each sample was processed and hybridized to SurePrint G3 Human Gene Expression Microarray platform (G4858A; Agilent Technologies, Santa Clara, USA) according to manufacturer’s protocol. Microarray slides were scanned using Agilent G4900DA Sure Scan Microarray Scanner (Agilent Technologies, Santa Clara, USA). After scanning the image data files were imported into the GeneSpring GX gene expression software, Version 12.6 (Agilent Technologies, USA) for detailed analysis.

Total RNA extraction was performed using the PicoPure RNA Isolation kit, (Life Technologies Inc., Burlington, ON) under an RNase-free environment and including a DNase digestion (Qiagen, Toronto, ON) step. RNA quality and concentration were verified with a 2100 Bioanalyzer (Agilent, Santa Clara, CA), using RNA 6000 Nano reagents (Agilent). All hybridized samples had a RIN between 7.0 and 9.3. Using 5 ng of extracted total RNA as starting material, linear amplification of the mRNA fraction was performed using the RiboAmp HS^Plus RNA Amplification Kit (Life Technologies Inc., Burlington, ON) which relies on T7 RNA polymerase in vitro transcription (IVT) to yield antisense RNA (aRNA).

Under an RNase-free environment, human blood total RNA was isolated using a Ribopure RNA isolation kit (Thermo Fisher Scientific, Waltham, MA). RNA concentration and quality were verified with an Agilent Bioanalyzer (Agilent Tecknologies, Santa Clara, CA), using RNA6000 Nano reagents (Agilent Technologies, Santa Clara, CA). Purified RNA was used for quantitative Real-Time PCR in our laboratory and for microarray assays performed at GenUs BioSystems (Northbrook, IL).

Bacterial RNA was extracted using the Qiagen RNeasy Kit Mini (Qiagen, Hilden, Germany) according to the manufacturer’s instructions with minor modifications. Briefly, precipitated cells from a 45 mL culture were resolved in 200 μL TE buffer and 15 mg/mL lysozyme, incubated for 30 min at room temperature and vigorously mixed every 2 min for 10 s for enzymatically disruption of the cells. Approximately 700 μL of RLT buffer and one spatula of glass beads were added and mixed vigorously for 3 min for mechanical disruption of the cells. Samples were centrifuged (3 min, 10,000 rpm, 4°C) and the supernatant was mixed with 470 μL of 100% ethanol. Afterwards, RNA purification was carried out using the Qiagen RNeasy Kit protocol. DNA contaminating the RNA samples was removed using RNase-free DNase I twice (Qiagen, Hilden, Germany). The RNA was further assessed by analysis on the Bioanalyzer 2100 (Agilent, Santa Clara, CA, United States) and RNA 6000 Nano Reagents (Agilent, Santa Clara, CA, United States). According to the amount of RNA extracted and its quality [RNA integrity numbers (RINs) > 8], the best samples per time point were used for transcriptomic analysis.

Total RNA was isolated using the RNeasy minikit (Qiagen) (liver) or Qiazol followed by the RNeasy (muscle, fat). RNA quality was verified using a 2100 Bioanalyzer with RNA 6000Nano Reagents (Agilent Technologies).
Library preparation and rRNA depletion was performed using the Illumina TruSeq stranded/unstranded mRNA Library Prep Kit v2 chemistry in an automated system (Agilent Bravo liquid handling platform) starting with 1 μg total RNA for each sample. Libraries were sequenced on the Illumina HiSeq2500 or HiSeq4000. Sequencing quality was assessed with FastQC (http://www.bioinformatics.babraham.ac.uk/ projects/fastqc/). Reads were mapped to the mouse genome mm10 (Ensembl build 38.91) and reads per gene were counted using STAR version 2.4.2a⁴⁹ . Gene count normalization and differential expression analysis was performed using DESeq2^{50 (link)}. For gene annotation, biomaRt was used^{51 (link)}. Functional enrichment according to gene ontology was carried out using GOrilla^{52 (link)}. All scripts are deposited at github (https://github.com/FranziG/E47KO-liver). Sequencing data, normalized count data and DESeq2 output can be accessed via NCBI’s Gene Expression Omnibus using the accession number GSE111877.