Invitrap spin universal rna mini kit

Total cellular RNA was extracted using the InviTrap® Spin Universal RNA Mini Kit (Stratec Biomedical, Germany) according to the manufacturers’ instructions and as previously described (Shytaj et al, 2020 (link)). RNA concentration was assessed using a P‐class P 300 NanoPhotometer (Implen GmbH, Munich, Germany).

RNA was isolated from the frozen whole blood samples (−80°C), in accordance with the manufacturer's protocol using TRI Reagent® (Sigma-Aldrich, USA). The aqueous phase was purified in accordance with the manufacturer's protocol using an InviTrap Spin Universal RNA Mini Kit (Stratec Biomedical Systems, Germany). The purity and quantity of isolated RNA were assessed using a Synergy HTX Multi-Mode Microplate Reader equipped with a Take3 Micro-Volume Plate and connected to a PC running Gen5 Software (BioTek Instruments Inc., Winooski, VT, USA). Isolated RNA (20 ng/μL) was transcribed onto cDNA with a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems™, Waltham, MA, USA). Quantitative assays were executed using a TaqMan Hs01075529_m1 probe for human NOS2 genes and an Hs02786624_g1 for endogenous control, which was GAPDH (Life Technologies). Reactions were carried out using a TaqMan Universal Master Mix II, without UNG (Life Technologies) in a BioRad CFX96 real-time PCR system (BioRad Laboratories, Hercules, CA, USA), all in accordance with the manufacturers' protocols. Relative expression of NOS2 was obtained using the equation 2^−ΔCt, where ΔCt is the threshold cycle (Ct) value for the target gene minus Ct values obtained for the housekeeping gene GAPDH [17 (link)].

Peripheral blood mononuclear cells (PBMCs) were extracted from venous EDTA blood using Ficoll-Paque PLUS (GE Healthcare) as previously described [18 (link)]. From the PBMCs, positive separation with MACS CD8 antibody MicroBeads (Cat. No. 130-045-201, Miltenyi Biotec, Bergisch Gladbach, Germany) was performed using an OctoMACS magnetic separator (Miltenyi Biotec) following the manufacturers protocol. From the separated cell populations, DNA and RNA were extracted using the InviTrap Spin Universal RNA Mini Kit for the amplification and library preparation in batches 1–3 (Stratec Biomedical, Birkenfeld, Germany) according to manufacturer’s instructions. From the blood donor samples, DNA was extracted with Nucleospin Tissue DNA extraction kit (Machery Nagel, cat. no 740952.250) according to the manufacturer’s instructions. To obtain better yield of DNA the QIAamp DNA Mini Kit (Qiagen) was used for batch 4. The purities of the separated CD8+ cells were tested in 52% of the 197 samples by flow cytometric analysis, in which T cells (CD3+) represented 89–99% of the cells and the observed purities for CD8 were all ≥ 84%. The CD8 subpopulations (CD27 and CD45RA positives and negatives) were not tested for purities, because of the small number of cells left after sequential negative and positive selection with immunomagnetic beads.

Total RNA was isolated with the InviTrap Spin Universal RNA Mini Kit (Stratec). Random hexamer primer and AMV Reverse Transcriptase (NEB) were used for reverse transcription. Quantitative PCR reactions were carried out using the LightCycler480 (Roche) with Power SYBR Green PCR Master Mix (Thermo Fisher Scientific). Using GAPDH or Actin as a reference gene, the relative expression levels compared to the control were calculated by the ΔΔCp method. Primer sequences are listed in Supplementary Table 5.

RNA-Seq was performed as described earlier [78 (link)]. Briefly, RNA was isolated from whole-cell lysates using InviTrap Spin Universal RNA Mini Kit (Stratec) according to the manufacturer’s instructions. For library preparation, 1 μg of RNA was poly(A) selected, fragmented, and reverse transcribed with the Elute, Prime, Fragment Mix (Illumina). End repair, A-tailing, adaptor ligation, and library enrichment were performed as described in the Low Throughput protocol of the TruSeq RNA Sample Prep Guide (Illumina). RNA libraries were assessed for quality and quantity with the Agilent 2100 BioAnalyzer and the Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies). RNA libraries were sequenced as 100 bp paired-end runs on an Illumina HiSeq4000 platform.