Ribominus eukaryote system v2

Gene expression datasets were generated by RNA sequencing of PBMCs samples obtained from the study participants as described in our previous papers. Briefly, PBMCs specimens were isolated from whole blood samples using density gradient centrifugation with Gradisol L reagent (Aqua-Med, Łódź, Poland). A diversity of white blood cells subpopulations in studied groups were evaluated using the whole blood morphology analysis (Figure S5). Total RNA was isolated from PBMCs samples using TRI Reagent Solution (Applied Biosystems, Foster City, CA, USA). Total RNA samples underwent ribodepletion procedure using RiboMinus Eukaryote System v2 (Ambion, Austin, TX, USA) and were subjected to whole transcriptome libraries preparation using Ion Total RNA-Seq Kit v2, Magnetic Bead Cleanup Module kit and Ion Xpress RNA-Seq Barcode 01-16 Kit (Life Technologies, Carlsbad, CA, USA). Libraries were sequenced on Ion 540 chips (Life Technologies) using Ion S5 XL System (Thermo Fisher Scientific, Waltham, MA, USA). Raw sequences were aligned to 55,765 genes of hg19 human genome using Torrent Suite Software v5.0.4. and Ion Torrent RNASeqAnalysis plugin v.5.0.3.0 (Thermo Fisher Scientific). Statistics of parameters describing transcriptome libraries and primary results of sequencing data analysis are provided in Table S2.

Total RNA was extracted with Trizol Reagent (Invitrogen Ambion) and treated with a RQ1 RNase-free DNase (Promega). One μg of RNA was used to generate cDNA with miScript II RT kit (Qiagen). Quantitative real-time PCR was performed in triplicate using SYBR Green ROX mix (Thermo Scientific) on an Applied Biosystems 7900HT. The normalized expression levels were calculated according to the ΔΔCt method. The snRNA, 5 S, 5.8 S, RPL13a and SDHA primers have been reported (Sapaly et al., 2018 (link)). The RNA sequencing approach was carried out at the Genom’ic Core Facility at the Institut Cochin, University of Paris Descartes. Briefly, ≈1 μg of total RNA with RNA Integrity Number > 8 (Bioanalyzer RNA nano chip, Agilent) isolated from cells treated with flunarizine (n = 3) and DMSO (n = 1) was used for rRNA depletion with the low Input RiboMinus Eukaryote System v2 (Ambion, Life technologies). The depleted RNAs were used to generate cDNA libraries according to the manufacturer’s protocol (Ion total RNA-Seq kit V2, Thermo Fisher Scientific). The sequencing was performed on Ion Chef (Life technologies). After quality control of the run and adaptor trimming, the reads were mapped to a reference genome using the STAR aligner. Differentially expressed genes and transcript levels were determined with the DESeq2 algorithm.

RNA extraction was performed using the TRIZOL™LS reagent (Invitrogen, Carlsbad, CA, USA). rRNA depletion was performed using the RiboMinus™ Eukaryote System v2 (Ambion^®) following standard protocols. First, total RNA was hybridized with biotinylated RiboMinus™ Eukaryote Probe Mix v2. Next, the rRNA-probe complexes were removed from the total RNA by captured with streptavidin-conjugated RiboMinus™ Magnetic Beads. The resulting rRNA-depleted RNA was concentrated and purified with Nucleic Acid Binding Beads. The cDNA libraries were barcoded using the ion total RNA-Seq Kit v2 (Ambion^®) following standard protocols. RNA sequencing was performed on the Ion Proton™ System (Life Technologies, Carlsbad, CA, USA). Data analysis was performed using Torrent Suite™ Software 4.0 (Ion Torrent). Genes with twofolds and more than twofolds up or down change in LPS-stimulated A549 cells were thought to be significant, compared with the control group.