Sybr green mix

All NAc tissues were collected at 2hr after CPP test. Total RNA and DNA were extracted using DNA/RNA isolation kit (Tiangen Biotech, Beijing, China) following the manufacturer’s instructions, and 1.0 μg total RNA was reverse-transcribed into single-strand cDNA using Superscript III (Invitrogen, Carlsbad, CA, USA). Real-time PCR was performed on an Opticon 2 real-time PCR machine (Rad, Hercules, CA, USA) using SYBR Green mix (Tiangen) with the following cycling program: 95 °C for 10 min, 40 cycles of 95 °C for 25 s, 60 °C for 25 s, and 72 °C for 25 s. An aliquot of cDNA was amplified with using a pair of primers as shown in Table 1. GAPDH was used as an internal control for normalization. Each sample was tested in triplicates.

Gene expression of senescence markers (p16 and p53) was analyzed by real-time PCR analysis. Briefly, total RNA from the P6 human disc NP cells was extracted using TRizol reagent (Invitrogen, U.S.A.) and synthesized into cDNA using a BeyoRT™ II First Strand cDNA Synthesis Kit (Beyotime, China) according to the manufacturer’s instructions. Then, the PCR process was performed on a reaction system consisting of cDNA, gene primers, and SYBR Green Mix (TIANGEN, Beijing, China). β-actin was used as a reference gene. The PCR protocol is: 95°C for 3 min, followed by 35 cycles of 95°C for 10 s, 56°C for 15 s, and 72°C for 30 s. The primers (Table 1) were purchased from a domestic bio-company (Shanghai Shenggong, China). The relative gene expression was calculated according to the method of 2^―ΔΔC_t.

These procedures were performed as previously described^{20 (link),23 (link)}. Briefly, total RNA was isolated using TRIzol^® Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions. Reverse transcription for first-strand cDNA was synthesized using the RevertAid^TM First Strand cDNA Synthesis Kit (cat. no. 6210A, TaKaRa Bio, Inc., Otsu, Japan). Subsequently, qPCR was performed in a CFX96 Real-Time PCR Detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using SYBR^® Green mix (Tiangen Biotech Co., Ltd., Beijing, China). The qPCR thermal cycling conditions were as follows: a denaturation step at 95 °C for 15 min and 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s and elongation at 72 °C for 30 s. The amplified products were examined using the 2^−∆∆Cq method^{24 (link)}, and each sample was calibrated to the expression levels of the housekeeping gene GAPDH. The primers are shown in Supplementary Table 1.

For Real-time quantitative reverse transcription PCR (Real-time PCR) analyses, the first-strand cDNA was synthesized using TransScript® First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). Real-time PCR was performed using SYBR Green Mix (TIANGEN, Beijing, China) in a LightCycler96 instrument (Roche, Switzerland), with the maize GAPDH gene as the internal reference. The ABA signaling pathway genes ZmSnRK2.4, ZmPP2C-A4, ZmPP2C-A7, ZmRD17, ZmRAB18, and ZmLEA were selected for expression validation using specific primers [25 (link)].

The extraction of total RNA was completed according to the manufacturers’ method described by the RNAprep Pure Plant Kit (TIANGEN Biotech, Hainan, China). In this experiment, containers and consumables were treated with RNase inactivation to ensure the integrity of the extracted RNA. The next steps were required to be completed on ice to confirm that the samples were not degraded. The RNA was subjected to cDNA synthesis with Fastking One Step RT-PCR Kit (TIANGEN Biotech, Hainan, China) following the instructions of the reagent manufacturer. qRT-PCR was conducted to examine the mRNA levels of the genes. In total, 1 μL of cDNA, together with 5 μL of SYBR green mix (TIANGEN Biotech, Hainan, China), 0.3 μL each forward and reverse primer, were added into the PCR mixtures (with the total volume of 10 μL). The PCR reactions were performed in A CFX Connect Detection System (Bio-Rad, CA, USA). Actin was selected as the reference gene. The method of 2^–ΔΔCt was adopted for calculation of results. The primer sequences were provided in Supplementary Table 1.

The real time PCR (qPCR) was used for verifying the significantly up or down regulated genes which were given out by the RNA-Seq analysis as described above. We tested all 9 rhythm genes by qPCR (Supplementary Materials Table S1 showed the primer of genes). The analysis was performed on an Opticon 2 RT-PCR machine (Bio-Rad, Hercules, CA, USA) using SYBR Green mix (Tiangen, Beijing, China) with the following cycling program: 95 °C for 10 min, 40 cycles of 95 °C for 25 s, and 60 °C for 1 min. Glyceraldehyde 3-phostaphate dehydrogenase (GAPDH) was used as an internal control for normalization, and each sample was run in triplicate. The aliquot of cDNA was amplified with a pair of primers for each.

These procedures were performed as previously described^{5 (link),17 (link)}. Briefly, total RNA was isolated using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions. First-strand cDNA was synthesized via reverse transcription using the Revert Aid^TM First Strand cDNA Synthesis Kit (cat. no. 6210A; TaKaRa Bio, Inc., Otsu, Japan). Subsequently, qRT-PCR was performed using a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and SYBR^® Green mix (Tiangen Biotech Co., Ltd., Beijing, China). The qRT-PCR thermal cycling conditions were initiated using a denaturation step at 95 °C for 15 min and comprised 40 cycles (denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s and elongation at 72 °C for 30 s). The amplification products were analyzed using the 2^−∆∆Cq method^{18 (link)}, and the expression levels in each sample were calibrated to those of the housekeeping gene GAPDH. The primers employed for amplifying HDAC4, E-cadherin, N-cadherin, Snail, Slug, and GAPDH are listed in Supplementary Table 1.