Miscript cdna synthesis kit

To validate the expression of miRNAs and genes of interest, total RNAs isolated from pulmonary infiltrating mononuclear cells from the lungs of Naïve, OVA-veh, and OVA-res groups were used. The concentration and the quality of RNAs were analyzed using Nanodrop 2000 (Thermo Fisher Scientific, Rockford, IL, USA). Equal amounts of RNAs were used for cDNA synthesis. To examine the expression of miRNAs, cDNAs were synthesized using miScript cDNA Synthesis kit and following the protocol of the company (Qiagen, Valencia, MD, USA). SYBR Green PCR kit from Qiagen was used and the protocol of the company was followed. To detect gene expression, SSO- Advanced SYBR Green PCR kit (Bio-Rad, Hercules, CA) was used and the protocol of the company was followed. Real-Time PCR was performed for 39 cycles and the details are as follows: 30 s 98°C (denaturation step), 60 s at 60°C (annealing step) and 60 s at 72°C (extension step, followed by incubation for 10 min at 72°C. The gene expression was normalized to GAPDH. GAPDH housekeeping gene was used because its expression is reliable for one kind of cells 41 (link), 42 (link). The PCR results of miRNAs expression were normalized to Snord 96A (small nucleolar RNA, C/D box 96A) was used as a control to assess the level of miRNA (43 (link)). The details of miRNAs and primer sequences for genes used in qPCR are described in Supplementary Table 1.

Trizol reagent (Beyotime, Shanghai, China) were applied to extract the all the RNA of the cell or tissue fragment, then PrimeScript RT Reagent Kit (Takara, Tokyo, Japan) was employed for synthesis of cDNA under the instruction from manufacture. The miRNA was converted to cDNA using miScript cDNA synthesis kit (Qiagen, Dusseldorf, Germany) following the manufacturer’s protocol. Later, SYBR Select Master Mix and ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) were applied to carrying out qRT-PCR. β-actin and U6 were chosen as internal references for mRNA and miRNAs respectively. Relative expressions of mRNAs and miRNAs were quantified using 2^{^-ΔΔCt} method. Supplementary Table 1 displayed all the primer sequences.

Cytokines levels were measured in plasma by using Bio-Plexmultiplex immunoassay system (Bio-Rad,Hercules, CA), as described by us previously^{45 (link)}. RNA was isolated from epididymal fat pad using the E.Z.N.A.® Total RNA Kit (Omega Bio-Tek, Norcross, GA). The purity and concentration of the RNA were confirmed spectrophotometrically with Nanodrop (Thermo Scientific,Waltham, MA). Total RNA was converted to cDNA using the miScript cDNA synthesis kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. SsoAdvanced™ Universal SYBR® Green Supermix kit (Bio-Rad,Hercules, CA) was used to analyze gene expression, and GAPDH was used as the housekeeping gene. List of all the primers has been provided in Supplementary Table 5. Complete Blood Cell count (CBC) was performed using hematological analyzer VetScan HM5 (ABAXIS, Union City, CA). Circulating LPS level was quantified as previously described^{46 (link)}. Colonic myeloperoxidase was assessed according to the manufacturer’s instruction (Abcam, Cambridge, MA)^{47 (link)}. Free fatty acid was quantified in serum according to the manufacturer’s instruction (Zen-Bio Inc, Research Triangle Park, NC).