Prism software version 7

In vitro experiments were routinely repeated at least three times unless otherwise noted. Data are expressed as mean ± SEM unless otherwise indicated. Statistical differences were measured using an unpaired two-sided Student’s t-test, one-way ANOVA with Bonferroni correction for multiple comparisons or log-rank test when appropriate. Normality was checked using the Kolmogorov-Smirnov test. A non-parametric test (Mann-Whitney) was used when data did not pass the normality test. A value of p ≤ 0.05 was considered statistically significant. Data analysis was performed using GraphPad Prism Software Version 7 (GraphPad, San Diego, CA).

RNA was extracted using RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacture's instruction. cDNA was synthesized using Ready-To-Go T-primed first strand kit (Invitrogen) or OmniScript Reverse Transcriptase Kit (Qiagen) or Biozym cDNA synthesis kit including random hexamers (Biozym Scientific GmbH, Oldendorf, Germany). Quantitative real-time PCR was performed with StepOne™ Real-Time PCR System (Applied Biosystem, Darmstadt, Germany). In each reaction, 2 μl cDNA, 0.4 μl reverse and forward primers, 5 μl SYBR Green mix (Bioline, Taunton, USA) and 2.2 μl DEPC-H₂O were mixed in one well in a 96-well plate and centrifuged briefly. After the initial denaturation step at 95 °C for 15 min, PCR reaction was cycled for 40 times with denaturation at 95 °C for 30 s and annealing-extension temperature at 65 for 30 s. Relative expression was calculated following 2-ΔΔCT method (Livak and Schmittgen, 2001 (link)). Primers are listed in Table S1. Statistical analysis was done using REST software and if p < 0.05, it is reported as statistically different (Pfaffl et al., 2002 (link)). The graphs were generated using GraphPad Prism Software version 7 (GraphPad Software Inc., California/USA).

The categorical variables are expressed as number and percentage and the continuous variables as median and interquartile range (IQ). The Mantel-Haenzsel chi-square test for trend was used to assess whether there was a statistically significant linear trend in the proportion of infections due to the most frequent microorganisms over the study period. For analyses, a p-value ≤ 0.05 was considered statistically significant. Statistical analyses were performed using IBM^® SPSS Statistics v 20.0 software (SPSS Inc., Chicago, IL, USA) and GraphPad PrismSoftware, version 7 (GraphPad Software, La Jolla, CA, USA).

The number of animals used in each study is listed in the figure legends. Data are expressed as average ±SEM. Statistical differences were measured using unpaired 2-sided Student t-tests and 2-way ANOVAs with Bonferroni correction for multiple comparisons. Normality was checked using the Kolmogorov-Smirnov test. A value of P ≤ 0.05 was considered statistically significant. Data analysis was performed using GraphPad Prism Software Version 7 (GraphPad). Metabolic cage data was analyzed using the CalRapp program. Differences in the relationship between respiratory parameters and body weight were analyzed using general linear model analysis of covariance (GLM ANCOVA), while RER and activity were analyzed by ANOVA. Specific statistics used for scRNA-seq and downstream analysis is detailed in Single-Cell RNA-seq data analysis section.

For genetic association studies, statistical comparisons were carried out using an online SNP stats program [19 (link)] or SPSS version 17 as described earlier [20 (link)]. The strength of association was represented in terms of odds ratio (OR) with 95% confidence intervals (CI). The OR with 95% CI was adjusted for age and gender. For cytokine levels and their association with genotypes or disease severity, the Mann–Whitney U test was used to compare two groups while the Kruskal–Wallis test with multiple corrections was used to compare multiple groups. Receiver operating curve analysis was performed to assess the utility of cytokines as biomarkers in predicting fatal outcomes. Graphpad prism software version 7 was used for the analysis.

Flow cytometry data were analyzed using FlowJo software, version 7.6.4 (Tree StarInc., Ashland, OR). The levels of significance of the differences between groups were examined by, either the Mann-Whitney test (unpaired samples) or the Wilcoxon matched-pair test (paired samples), as nonparametric data were obtained. The correlation between different measurements of immune response was analyzed using Spearman rank test, after graph analyses. These analyses were performed with the GraphPad-Prism software, version 7 (GraphPad Software, Inc., USA).