Sds 2

qRT-PCR was performed on RNA samples prepared as described above. The cDNA was produced by reverse-transcribing 400 ng of total RNA using a mix of random hexamers and oligo d(T) primers and Primescript reverse transcriptase enzyme (Takara Bio, San Jose, CA, USA). The efficiency of each pair of primers was tested with serial dilutions of cDNA. Oligonucleotides are indicated in Table 2. PCR reactions (10 µL volume) contained 1:20 diluted cDNA, 2 × Power SYBR Green Master Mix (Applied Biosystems by ThermoFisher, Waltham, MA, USA), and 300 nM of forward and reverse primers. PCRs were performed on a SDS 7900 HT instrument (Applied Biosystems by ThermoFisher, Waltham, MA, USA) with the following parameters: 50 °C for two minutes, 95 °C for ten minutes, and 45 cycles of 95 °C 15 s, 60 °C one minute. Each reaction was performed in three replicates on 384-well plate. Raw Ct values obtained with SDS 2.2 (Applied Biosystems by Thermo Fisher, Waltham, MA, USA) were imported into Excel and normalization factors were calculated using the GeNorm method as described by Vandesompele et al. [34 (link)].

To determine IL36R expression, single-cell suspensions were made from spleens and lymph nodes of C57BL/6 mice. Naive CD4⁺T (CD44_low CD62L_high), CD8⁺ T, Treg (CD4⁺CD25⁺) cells were purified by fluorescence-activated cell sorting (FACS). Total RNA was extracted using the TrIzol reagent (Invitrogen Life Technologies) according to the manufacturer's protocol. Total RNA was reverse transcribed using SuperScript II Reverse transcriptase (Invitrogen Life Technologies). The mRNA levels for genes of interest were examined by quantitative RT-PCR using SYBR Green PCR Master Mix (Applied Biosystems). Values obtained with the SDS 2.2 (Applied Biosystems) were imported into Microsoft Excel for analyses and gene expression was calculated using the comparative method (2^−δCt) for relative quantification by normalization to GAPDH gene expression.

Total RNA was extracted from lungs of M. bovis BCG and M. tuberculosis infected mice, as well as after LPS challenge using TRIzol reagent, according to manufacturer’s instructions, and further purified on RNeasy columns (Qiagen AG, Hombrechtikon, Switzerland). Total RNA (1 μg) was reverse transcribed using SuperScript II Reverse transcriptase (Invitrogen Life Technologies). The mRNA levels for genes of interest were examined by quantitative RT-PCR using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). Values obtained with the SDS 2.2 (Applied Biosystems, Zug, Switzerland) were imported into Microsoft Excel for analyses and gene expression was calculated using the comparative method (2^–ΔCt) for relative quantification by normalization to Gapdh or L32 gene expression. The primer sequences used for real-time quantitative RT-PCR are described in Table 1.

RNA extraction (myoblasts, 48 h-MuRC and 96 h-MuRC) was performed with TRIzol reagent (Life Technologies) according to the manufacturer's protocol. After measuring sample concentrations, 0.5 µg of total RNA was reverse-transcribed with the PrimeScript™ RT reagent Kit (TaKaRa) according to the manufacturer's instructions. qRT-PCR experiments were all performed at the iGE3 Genomics Platform of the University of Geneva (http://www.ige3.unige.ch/genomics-platform.php) on an SDS 7900 HT instrument (Applied Biosystems). Raw threshold-cycle (Ct) values obtained with SDS 2.2 (Applied Biosystems) were imported into Excel. Normalization factor were calculated using the geNorm method [34 (link)]. Primers used for qRT-PCR were: Hs β2-microglobulin forward, 5′-TGCTCGCGCTACTCTCTCTTT-3′; Hs β2-microglobulin reverse, 5′-TCTGCTGGATGACGTGAGTAAAC-3′; Hs EEF1A1 forward, 5′-AGCAAAAATGACCCACCAATG-3′; Hs EEF1A1 reverse, 5′-GGCCTGGATGGTTCAGGTA-3′; Hs Pax7 forward, 5′- AAACACAGCATCGACGGCA-3′; Hs Pax7 reverse, 5′- CTCGTCCAGCCGGTTCC-3′; Hs MyoD forward, 5′- TGCCACAACGGACGACTTC -3′; Hs MyoD reverse, 5′- CGGGTCCAGGTCTTCCGAA-3′; Hs HES1 forward, 5′- CAGATGACGGCTGCGCT -3′; Hs HES1 reverse, 5′- TCGGTACTTCCCCAGCACAC-3′.