Sepasol reagent

Total RNA was extracted from livers using Sepasol reagent (Nacalai Tesque, Kyoto, Japan) and was reverse-transcribed using the PrimeScript RT Master kit (Takara Bio Inc., Shiga, Japan) according to the manufacturer’s protocols. qPCR was performed using SYBR Premix Ex Taq (Takara Bio Inc.) and specific primer sets with the Thermal Cycler Dice Real Time System Single (Takara Bio Inc.). Primer sequences for Acat2, Nr1h3, Ces3a, Ces3b, Nr1h4, Nr5a2, Nr0b2, Cyp7a1, Cyp8b1, Cyp27a1, Cyp7b1, Slc10a1, Slco1a1, Abcb11, Abcc2, Abcc1, Abcc3, Abcc4, Slc51b, Cd14 and Cybb are summarized in Supplementary Table S2 online. Other qPCR primers used have been described previously16 (link)23 (link)49 (link). The expression levels of mRNA were normalized to those of peptidylprolyl isomerase A (Ppia) mRNA.

Total RNA was extracted from Hepa1-6 cells using Sepasol reagent (#09379-84, Nacalai Tesque, Kyoto, Japan) and 1 μg of total RNA was used for the first-strand cDNA synthesis using ReverTra Ace (#TRT-101, Toyobo, Osaka, Japan) and Random Primer (#48190011, Thermo Fisher Scientific). The reaction profile was 30 °C for 10 min, 42 °C for 60 min, and 99 °C for 5 min. Then, 100 ng of cDNA was subjected to quantitative RT-PCR using a StepOnePlus Real-time PCR System (Applied Biosystems, Foster City, CA, USA) with Fast SYBR Green Master Mix Reagent (#4385612 Thermo Fisher Scientific). The primer sets are listed in Supplementary Table 1b. The reaction profile was 95 °C for 20 s followed by 40 cycles of 95 °C for 30 s and 60 °C for 30 s. The mRNA levels were normalized to those of 36B4 and analyzed using the comparative CT method.
For the analysis of the copy number after random integration of the dCas9 cassette, 30 ng of genomic DNA were analyzed by quantitative real-time PCR using the comparative CT method. Quantification of the dCas9 DNA was normalized to that of the single-copy nuclear gene Ndufv1 (GenBank accession no. NM_133666) using the primer pairs, as described in previous reports^{39 (link),40 (link)}. PCR primers for dCas9 and Ndufv1 were as described in Supplementary Table 1c.

Total RNA of the PVAT was isolated using Sepasol reagent (Nacalai Tesque, Inc.). RNA was reverse transcribed with Random Primer (Thermo Fisher Scientific Inc., Waltham, MA, USA) and ReverTra Ace (Toyobo Co., Ltd., Osaka, Japan). Quantitative RT-PCR was performed using StepOnePlus Real-time PCR System with Fast SYBR Green Master Mix Reagent (Thermo Fisher Scientific Inc.). Primers are listed in S Table. Data were normalized to the 36b4 levels, and analyzed by the comparative CT method.

Total RNA of the liver and epididymal fat was isolated using Sepasol reagent (Nacalai Tesque, Inc.) and RNeasy Plus Universal Mini Kit (Qiagen, Hilden, Germany), respectively. RNA was reverse transcribed with Random Primer (Thermo Fisher Scientific Inc., Waltham, MA, USA) and ReverTra Ace (Toyobo Co., Ltd., Osaka, Japan). Quantitative RT-PCR was performed using StepOnePlus Real-time PCR System with Fast SYBR Green Master Mix Reagent (Thermo Fisher Scientific Inc.). Primers are listed in S1 Table. Data were normalized to the 36b4 levels, and analyzed by the comparative CT method.

The harvest of the total renal RNA was conducted by utilizing Sepasol reagent (Nacalai Tesque, Inc., Kyoto, Japan). Then, by utilizing ReverTra Ace (Toyobo Co., Ltd., Osaka, Japan) and Random Primer (Thermo Fisher Scientific, Inc., Waltham, MA, USA), cDNA first-strand was carried out. To perform the steps of the quantitative real-time polymerase chain reaction (qRT-PCR), Step One Plus Real-Time PCR with Fast SYBR Green Master Mix Reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) were utilized. Caspase-3, Keap1, NF-κB, Nrf2, PPARγ, HO-1, and β-actin primers (Table 1) were selected to be assessed, purchased, and custom-made by (Thermo Fisher Scientific, Inc., Waltham, MA, USA). According to the Livak and Schmittgen method, the comparison CT technique was utilized for determination of the relative gene expression [44 (link)]. As an endogenous reference gene, β-actin was utilized.

Total RNA was extracted from the liver using a Sepasol reagent (Nacalai Tesque, Kyoto, Japan) and was reverse‐transcribed using PrimeScript RT Master Kit (Takara Bio Inc., Shiga, Japan), according to the manufacturers’ instructions. Afterward, quantitative real‐time PCR (qPCR) was conducted using a SYBR Premix Ex Taq (Takara Bio Inc. Shiga, Japan) and specific primer sets with a Thermal Cycler Dice Real‐Time System Single (Takara Bio Inc. Shiga, Japan). The primer sequences for qPCR in this study are shown in Table S2. The mRNA expression levels were normalized to that of cyclophilin mRNA. Portions of the RNA samples were sequenced (RNA‐Seq) and analyzed via qPCR analyses. RNA‐Seq was conducted as described elsewhere [19 (link)]. Portions of the 100 ng of total RNA from the livers of the control, CDAHFD‐0.1, and CDAHFD‐0.6 groups (treated for 13 weeks, n = 3–4) were used for the library preparation. Sequencing libraries were generated using TruSeq RNA Library Preparation Kit v2 (Illumina Inc., San Diego, CA, USA). The principal component analysis (PCA), differential expression analysis, generation of heat maps with hierarchical clustering of samples, and features and functional annotation analyses using Ingenuity Pathway Analysis (IPA) software (Ingenuity Systems, Qiagen Co., Ltd, Redwood City, CA, USA) were conducted as previously described [19 (link)].