Quantstudio 6 pcr system

Total RNA was extracted by RNA Purification Kit (EZBioscience) on the basis of the manufacturer’s instructions. RNA was reverse transcribed to cDNA by the Reverse Transcription Master Mix (EZBioscience). Reverse transcription qPCR was conducted by the Quant Studio 6 PCR System (Thermo Fisher Scientific) and 2 × Color SYBR Green qPCR Master Mix (EZBioscience). The primers are listed in table S2. Fold change was assessed using the 2^−ΔΔCt method using β-actin as a reference gene.

Total RNA of tissues was isolated with TRIzol reagent (Aidlab Biotechnologies, Beijing, China). After lysing for 5 min, chloroform (Thermo Fisher Scientific) was added to the mixed solution. Then the supernatant was mixed with isopropyl alcohol and centrifuged for 10 min at 12,000 rpm and 4°C. After washing with 75% alcohol twice, the precipitated RNA was dissolved by DEPC-water and stored at −4°C. Reverse-transcription was performed, and the synthesized cDNAs were amplified and quantified using the QuantStudio 6 PCR system (Thermo Fisher Scientific). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. All primer sequences are shown in Supplementary Table 1. The calculation and quantification of gene expression were based on the 2^–ΔΔCt method.

Total RNA was extracted from leaves at different developmental stages, as described above, and cDNA was synthesized from 2 μg total RNA using a cDNA synthesis kit (Vazyme, Nanjing, China) in a total volume of 20 μL. A total of nine DEGs related to anthocyanin biosynthesis in S0–S2 were selected for qRT-PCR. Gene-specific primers were designed using Primer Premier Software v.5.0 (Premier Biosoft, Palo Alto, CA, USA) (Table S3). Ultra SYBR Mix (CWBIO, Beijing, China) and the QuantStudio 6 PCR system (Thermo Fisher Scientific, Waltham, MA, USA) was used for qRT-PCR. Furthermore, 50 μL reaction mixture contained 2 μL cDNA (1:50 dilution), 25 μL of 2× Ultra SYBR Mix (CWBIO, Beijing, China), and 1 µL of each primer (100 nM final concentration). The amplification conditions were as follows: 95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. A melting curve analysis (55–95 °C) was performed at 95 °C for 15 s, 60 °C for 1 min, 95 °C for 15 s, and 60 °C for 15 s to confirm the specificity of the PCR amplification. Actin gene was used as an internal control. Relative expression levels were calculated using the 2^−ΔΔCt method [66 (link)]. All experiments were performed using three independent samples from three independent biological replicates.

Citrate synthase activity assays to assess tissue mitochondrial function were performed as described previously^{33 (link)}. Protein lysate (8 μg) was used for the citrate synthase activity assay following the manufacturer’s protocol (Sigma CS070). Tissue samples were homogenized in tissue lysis buffer provided with the kits to obtain protein lysates. After centrifugation and removal of the lipid layer, protein concentration was determined using the BCA Protein Assay (Thermo Fisher Scientific Inc.).
For gene expression assays, total RNA was extracted using Trizol (Invitrogen), treated with DNase (ThermoScientific) and reverse transcribed to cDNA (AppliedBiosystems) according to manufacturer’s instructions. Gene expression, normalized to 36B4, was analyzed by quantitative real-time RT-PCR (Sybr Green, 384-well plates) using the QuantStudio 6 PCR System (ThermoFisher). Primer sequences are available upon request.
To perform immunoblot analysis, homogenized tissue was lysed in protein lysis buffer (Sigma) containing protease and phosphatase inhibitors. Standard western blotting was performed using rabbit polyclonal antibodies to Ucp1 (1:500; Abcam; ab23841) and GAPDH (1:3000; CellSignaling; #2118). HRP linked Goat anti-Rabbit (1:3000; Biorad; #170–6515) was used as secondary antibody. Proteins were detected by chemiluminescence (Roche) and images were acquired using FusionFx (Peqlab).

Total RNA was isolated from jejunal tissues with the RNeasy Mini kit (Qiagen) per the manufacturer’s protocol. Quality of mRNA was verified with a Nanodrop 2000 spectrophotometer (Thermo Fisher). 500 ng cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems), with RNase A included in the reaction, following the manufacturer’s protocol. qRT-PCR analysis was performed with 25 ng cDNA/well using Taqman Universal Master Mix II with UNG (Applied Biosystems), on a QuantStudio 6 PCR system (Thermo Fisher) for the following Taqman probes: Actb (Mm02619580_g1), Lgr5 (Mm00438890_m1), Olfm4 (Mm01320260_m1), Tnf (Mm00443258_m1), Il1β (Mm00434228_m1), Nos2 (Mm00440502_m1), Il13 (Mm00434204_m1), Ccl2 (Mm00441242_m1), Ccl7 (Mm00443113_m1). Signals were normalized to Actb for each sample, and relative fold changes were calculated via ΔΔCt analysis.

Total RNA was isolated using the TRI Reagent (Sigma‐Aldrich) according to the manufacturer's protocol and subjected to DNaseI treatment (Promega). Total RNA was used for cDNA synthesis with SuperScript III Reverse Transcriptase (ThermoFisher Scientific). Quantitative PCR was performed in QuantStudio 6 PCR system (ThermoFisher Scientific) with Luminaris HiGreen qPCR Master Mix‐low ROX (Thermo Fisher Scientific) and 0.3 μM primer mix. miRNA expression was measured with TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems) and SensiFAST Probe Hi‐ROX Kit (Bioline) using predesigned TaqMan probes (ThermoFisher Scientific). The fold changes in expression were calculated by the ΔΔCT method using snoRNA202 and U6 RNA as reference small RNAs in mouse and human cells, respectively. See Appendix Table S1 for primer sequences.