Recombinant dnase 1

Total skin tissue RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), as described previously [24 (link),40 (link)]. RNA concentration and quality were analyzed using a CFX96^TM Real-Time System (Bio-Rad, Hercules, CA, USA). The sample was treated with recombinant DNase I (DNA-free; Ambion, Austin, TX, USA) to remove contaminating DNA, and RNA was reverse-transcribed using a reagent High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. A total of 50 PCR cycles were performed as follows: 95 °C for 15 s, 60 °C for 20 s, and 72 °C for 30 s for denaturation, annealing, and extension, respectively. The primers used are listed in Table S1.

RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA), according to the method described in previous studies5,15. The RNA concentrations and quality were determined with a CFX96^TM Real-Time System (Bio-Rad, Hercules, CA, USA). To remove contaminating DNA, samples were treated with recombinant DNase I (DNA-free; Ambion, Austin, TX, USA). RNA was reverse transcribed using the reagent High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Briefly, the cDNA strand was first synthesized from the total RNA and then the mixture of the primers and the cDNA products was amplified by PCR. The conditions of PCR amplification were 58°C for 30 mins, 94°C for 2 mins, 35 cycles of 94°C for 15 sec, 60°C for 30 sec, 68°C for 1 min, and then 72°C for 5 mins. Finally, the PCR products were separated on 0.8% agarose gel. Analysis was carried out using a gel imaging system (Bio-Rad, Hercules, CA, USA). Expression levels of SREBP-1c, SCD1, ACC1, FAS, PPARγ, DGAT2, PPARα, ACO, CPT1 and Nrf2 were calculated as a percentage relative to the intact group using β-actin RNA as the internal control. The sequences of the PCR oligonucleotide primers are listed in Table 1.

Total RNA was extracted with TRIzol reagent (Invitrogen Life Technologies), according to the manufacturer’s instructions. Genomic DNA was removed by treatment with recombinant DNase I (Ambion Life Technologies), and RNA quality and quantity were determined with a nanodrop spectrophotometer. cDNA was synthesized from 200 ng RNA using the High Capacity RNA-to-cDNA kit from Applied Biosystems. For quantitative RT-PCR, the CCR8 mRNA level was determined using TaqMan Gene Expression assays and TaqMan Universal Master Mix II (Applied Biosystems), according to the manufacturer’s instructions. Reactions were performed using a ViiA7 real-time PCR system (Life Technologies) and were run in triplicate. Water (no template control) and mRNA without reverse transcriptase were used as negative controls. Relative quantification of CCR8 mRNA levels was performed using the comparative Ct method with β-actin as the reference gene and the equation 2^−ΔΔCt.

Expressions of mRNAs, acetyl-CoA carboxylase 1 (ACC1), AMP-activated protein kinase (AMPK)α1, and AMPKα2 were measured in the liver, and gene expressions of leptin, adiponectin, uncoupling protein (UCP)2, CCAAT-enhancer-binding protein (C/EBP)α, C/EBPβ, sterol-regulatory-element-binding protein 1c (SREBP1c), PPARα, PPARγ, and fatty acid synthase (FAS) were measured in the periovarian fat tissue as a predominant region of white adipose tissues, as described previously [24 (link),25 (link)]. Total RNAs (about 5 μg) were extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and the concentration and quality were determined by the CFX96^TM Real-Time System (Bio-Rad, Hercules, CA, USA). The contaminating DNA was removed using recombinant DNase I (Ambion, Austin, TX, USA). The RNA was reverse-transcribed using the reagent High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). The PCR conditions were as follows: 10 min at 94 °C, 39 cycles of 15 s at 94 °C, 20 s at 57 °C, and 30 s at 72 °C. The expression was analyzed by the ABI Step One Plus^TM Sequence Detection System (Applied Biosystems), and normalized to that of GAPDH using the comparative threshold cycle method [26 (link)]. The oligonucleotide primers used for the PCR are listed in Supplementary Table S1.

Binary complexes were formed by incubating 10 nM [γ³²P]ATP-labeled DNA fragments and increasing concentrations of E. coli σ⁷⁰-RNAP (Holoenzyme, Epicentre). Binding reactions were done in buffer B (25 mM HEPES pH 7.5, 0.1 mM EDTA, 5 mM DTT and 10% glycerol), 50 mM KCl and 0.5 mg/ml bovine serum albumin), for 30 min at 30°C. The reactions were subjected to DNase I digestion (0.3 U Recombinant DNase I, Ambion) and treated as previously described [90] (link). The samples were analyzed on a 6% denaturing polyacrylamide-8.3M urea gel. A+G Maxam Gilbert sequencing reactions of the same DNA fragments were loaded alongside the samples. The bands were visualized using Phosphor screen cassette and Typhoon scanner 9400 (GE Healthcare).

Corneas were removed from the eyes by excision. Corneal tissue was immediately homogenized in Trizol reagent (Invitrogen) and the total RNA was isolated using Direct-zol RNA Miniprep kit according to the recommended protocol (Zymo Research). To eliminate potential contamination with genomic DNA (gDNA) the samples were subjected to additional treatment with recombinant DNase I (Ambion) for 30 min at 37°C and the total RNA was re-purified over RNeasy mini columns (Qiagen). Quality and the RNA concentration in the samples were evaluated using the RNA chip bioanalyzer (Agilent Technologies). As an additional step to test RNA quality, we performed RT-PCR assays using a panel of primers for several ubiquitously expressed genes including Gapdh and G protein subunits (Gnb1, Gnai2). Samples were depleted of both cytoplasmic and mitochondrial ribosomal RNA using Ribo-Zero-Gold kit (Epicentre). Library preparation for next generation sequencing was performed using Illumina TrueSeq library preparation kit that allows for paired-end directional RNA sequencing. Next-generation sequencing of corneal RNA was performed at the Hussman Institute for Human Genomics core facility using the Illumina HiSeq 2000 platform.