Intron spanning primers

To assess the expression of activation-related gene transcripts, RNA was extracted from U937 cells according to the manufacturer’s recommendations. Electrophoresis in 1% agarose gel was used to assess RNA integrity, and concentration and purity by absorbance 268/280 nm in a Nanodrop 100 (Thermo, USA). cDNA was generated using 250 ng of total RNA, random primers, and the Transcriptor first strand cDNA synthesis kit (Roche Cat. 04379012001, Mannheim Germany). Real time PCR was conducted with LNA hydrolysis probes from the Universal Probe Library Roche (UPL) (Roche, Cat 04683633001, Mannheim Germany), and intron spanning primers from Sigma. TLR2 (NM_003264.3) F 5′-CGTTCTCTCAGGTGACTGCTC-3′, R‘3-TCTCCTTTGGATCCTGCTTG-’5, UPL probe 14; CASP3 (NM_004346.3) F 5′-CTGGTTTTCGGTGGGTGT-3′, R 3′-CCACTGAGTTTTCAGTGTTCTCC-5′ UPL probe 34; CD11B (NM_000632.3) F 5′-GGCATCCGCAAAGTGGTA-3′, R 3′-GGATCTTAAAGGCATTCTTTCG-5′ UPL probe 9; and GADPH (NM_002046.3) F 5′-AGCCACATCGCTCAGACAC-3′, R 3′-GCCCAATACGACCAAATCC-5′ UPL probe 60.
One μL of each cDNA was amplified with 400 nM of primers, 100 nM of UPL probe, with the LightCycler TaqMan^® Master (Roche, Cat. 04535286001) followed by 45 cycles of 95° 10 s, 60° 60 s, and 72° 1 s. Reference gene GAPDH was used for relative quantification, according to the 2^−ΔΔCt method, using the non-stimulated cells as calibrator sample.

RNA was extracted using the TRIzol RNA Isolation Reagents (Thermofisher) according to the manufacturer's instructions. Briefly 5 × 10⁵ cells were washed carefully with PBS and lysed using of Trizol followed by the addition of chloroform. Samples were vortexed thoroughly and left at room temperature for 3 min then centrifuged at 12 000 × g and 4°C for 15 min for phase separation. The upper aqueous phase containing RNA was carefully transferred to a new Eppendorf tube. Isopropanol was added with proper mixing and incubated at room temperature for 10 min, then centrifuged at 12 000 × g and 4°C for 10 min to precipitate RNA. RNA pellet was washed twice with 75% ethanol and resolved in RNase-free water. cDNA is reverse transcribed using the SuperScript™ III First-Strand Synthesis System (Thermofisher) and random priming with hexamers. RT-qPCR is performed using primaQUANT™ CYBR green kit (Steinbrenner Laborsysteme) and intron-spanning primers from Sigma (Supplementary Table S8). Melting Curves are measured using the Lightcycler 480 II from Roche. The gene expression is normalized onto the housekeeping genes and reflects the relative percentage of expression towards the housekeeping genes. The data are presented as the mean ± SD value in independently repeated experiments. One-way ANOVA with Dunnett's multiple comparison test was performed to compare all to the WT untreated.

CD45.2⁺CD11b⁺ FACS-sorted cells were lysed and RNA was isolated and quantified by spectrophotometry. cDNA was synthesized using random primers and Moloney murine leukemia virus Superscript Reverse Transcriptase (II-MMLV) (Invitrogen). Relative mRNA levels were determined with a Biorad CFX96 cycler using the Fast Start SYBR Green Kit (Invitrogen). Intron spanning primers (Sigma-Aldrich) were designed and tested in our lab and are provided upon request Data were analyzed with Bio-rad CFX manager version 1.6 (Bio-rad) and checked for correct amplification and dissociation of the PCR-products. PBGD and GUSB served as reference genes. mRNA expression was determined relative to PBGD expression using the formula 2^{(CT index – CT PBGD)}.