Magnetic bead cleanup module kit

The procedure of study material preparation and sequencing was conducted as previously described in [28 (link)].
Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood specimens using density gradient centrifugation with Gradisol L reagent (Aqua-Med, Łódź, Poland). Proportions of white blood cells subpopulations in AAA group were obtained from venous blood morphology analysis results and were presented in Figure S1.
Small RNA fractions (for miRNA expression analysis) were isolated from PBMCs specimens of twenty eight AAA patients and nineteen control subjects using MirVana microRNA Isolation Kit (Ambion, Austin, TX, USA).
Total RNA specimens (for transcriptome analysis) were isolated from PBMCs samples of seven randomly selected AAA patients and seven randomly selected controls using TRI Reagent Solution (Applied Biosystems, Foster City, CA, USA).
Small RNA and transcriptome libraries were prepared using Ion Total RNA-Seq Kit v2, Magnetic Bead Cleanup Module kit, Ion Xpress RNA-Seq Barcode 01-16 Kit and sequenced on Ion 540 chips (all Life Technologies, Carlsbad, CA, USA) using Ion S5 XL System (Thermo Fisher Scientific, Waltham, MA, USA). Raw sequences of small RNA and transcriptomic libraries were aligned to 2792 human miRNAs from miRBase v21 (http://www.mirbase.org) and to 55,765 genes of hg19 human genome, respectively.

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.