Sybr kit

Total RNA was isolated using TRIzol Reagent according to manufacturer's instruction. Quantitative RT-PCR (qRT-PCR) was performed with Takara SYBR kit using the primers sets in Table 1 as previously described [20 (link)]. The 2^−ΔΔCT method was utilized to analyze the fold increase.

The total RNA was isolated using Trizol reagent (Takara, Dalian, China, Cat no. 9108). RNase R treatment was processed at 37°C with RNase R (Epicenter, WI, USA, Cat no. RNR07250). Complementary DNA (cDNA) was then directly synthesized and reversed with PrimeScript RT Master Mix (Takara, Dalian, China, Cat no. RR036A). qRT-PCR was performed using the SYBR kit (Takara, Dalian, China, Cat no. RR820A) and analyzed via a StepOne Plus system (Life Technologies, Carlsbad, CA) [16 (link)]. GAPDH and U6 were used as control genes for circRNA and miRNA, respectively. The fold changes were calculated through relative quantification (2^−ΔΔCt). The primer sequences are provided in Table S2.

Intestine and cell samples were processed for total RNA extraction using TRIzol reagent (Takara, Japan). Genomic DNA was removed at 42 °C for 2 min using the PrimeScript™ RT reagent kit (Takara), and then cDNA was synthesized. The program of cDNA synthesis consisted of 37°C for 15 min and 85°C for 5 s. Relative gene expression was performed with quantitative real-time polymerase chain reaction (qRT-PCR) using SYBR kit (Takara) and calculated by the method according to a previous study (22 (link)). β-actin was used as the internal reference gene. The primers for qRT-PCR were listed in the Table 2.

ChIP chromatin was prepared as described above. Beads with bound immunocomplexes were washed, eluted, and reverse cross-linked at 65⁰C overnight, subjected for RNase (final; 40 μg/ml) and proteinase K (Final; 100 μg/ ml) treatment and DNA were extracted by phenol–chloroform method. Input was prepared from 5% of the sonicated chromatin. 2 μL input or ChIP sample from the recovered DNA suspension of 50 μL was used for performing the qPCR analysis. Specific primers were designed against the HIT2 peaks obtained from ChIP-sequencing experiment (primers are listed in Additional file 16: Table S14). The enrichment of specific regions of the genome were validated by qPCR reaction using ChIP DNA (20 ng), 300 nM forward/reverse primers using SYBR kit from TAKARA. The Ct values of the duplicates showed minimal variability, and Ct values were used for calculating the percent of input using the following formula: $$\text{Percent}\text{input}=100\times 2(\text{zdjusted}\text{input}-\text{Ct}(\text{IP})).$$

The total RNA was extracted from the epididymal WAT using an RNA extraction kit (Accurate Biotechnology, Hunan, China). The cDNA was synthesized by a TIANScript RT kit (Tiangen Biotech, Beijing, China). In strict accordance with the instructions of the SYBR kit (Takara, Otsu, Shiga, Japan), RT-qPCR was performed on the Techne Quantica RT-PCR detection platform (Techne, Staffordshire, UK). The PCR sequence of each primer is shown in Table 1.

An EASYspin Plus RNA kit (Aidlab, Gdansk, Poland) was used for total RNA isolation and RNase-free DNase I (Qiagen, Hilden, Germany) was applied for removing genomic DNA. SuperScript III (Invitrogen, Waltham, MA, USA) applied for cDNA synthesis, which was used for further qRT-PCR. The experiment was performed by using SYBR kit (TaKaRa Biotechnology, Shiga, Japan) and LightCycle 96 real-time PCR machine (Roche, Basel, Switzerland). The detailed process and data analysis were developed as described previously by De Oliveira et al. (2012) (link) and Liu et al. (2018) (link)

With the help of TRIzol total RNA extraction reagent (No. 9108, Takara, Japan), total RNA was extracted from hepatopancreas. The amount and concentration of total RNA were determined spectrophotometrically with the aid of Nano-300 (No. 1053, ALLSHENG, China). The RNA solution was stored in DEPC water, which is ultrapure water treated with diethyl pyrocarbonate and autoclaved, free of impurities of RNA, DNA, and protein. The next step is to reverse transcribe the RNA to cDNA and store it at -20°C in preparation for subsequent real-time quantitative PCR (RT-qPCR). RT-qPCR amplification reactions were performed using the SYBR kit (RR420A, Takara, Japan). Primer sequences of target genes are shown in Table 2.