Sybr premix

Total RNA was extracted from liver tissues, and cDNA was synthesized. Next, gene expression was compared by quantitative reverse transcription (RT)-PCR for F9, alpha-L-iduronidas, iduronate 2-sulfatase, galactosidase-alpha, GBA, albumin, and Apoc3. Quantitative RT-PCR was conducted in quadruplicate with primers obtained from the Primer Bank (https://pga.mgh.harvard.edu/primerbank/) using SYBR premix (ThermoFisher Scientific), and gene expression levels were normalized to that of GAPDH. The sequences of the primers used are listed in Table S5. The AAV genome copy number in liver samples was determined using an AAVpro titration kit (Takara, Shiga, Japan) according to the manufacturer’s instructions. The number of AAV genome copies was determined against a standard curve. A conversion factor of ∼1.6 × 10² diploid cells per 1 μg mouse genomic DNA was used to calculate the number of diploid cells.

qRT-PCR was performed to assess CCKAR expression in fresh NSCLC tissues and para-tumor tissues. The total RNA was extracted by TRIzol agent (Thermo Fisher, Waltham, MA, USA). Reverse transcription of cDNA was accomplished with ReverTra kit (TOYOBO, Japan), and the quantitative real-time polymerase chain reaction (qRT-PCR) was applied with Thermo Fisher 7500 PCR System and SYBR Premix. The quantification of qRT-PCR results was analyzed by the 2^-ΔΔCt method with GAPDH as an internal control. The primers of CCKAR and GAPDH were designed as follows: CCKAR, forward: 5’-ATGGATGTGGTTGACAGCCTT-3’, reverse: 5’-AAGCGTCTCATTTTCGAGCCC-3’; GAPDH: forward: 5’-GAGTCAACGGATTTGGTCGT-3’, reverse: 5’-GACAAGCTTCCCGTTCTCAG-3’.

Total RNA was extracted from seedling shoots using Trizol reagent (Thermo Fisher Scientific). The extracted total RNA was treated with RQ1 RNase-free DNase (Promega, Madison, WI, USA), after which first-strand cDNA was amplified using M-MLV Reverse Transcriptase (Promega). The RT-qPCR was completed using an ABI 7500 Real-Time PCR System (Thermo Fisher Scientific) and SYBR Premix (Thermo Fisher Scientific). Two independent experiments were performed, each with three technical replicates. The relative expression was calculated as previously described^{27 (link)}.The results of a representative experiment are provided, with the data presented as the mean ± SD (n = 3).

qPCR analysis was performed for 15 DEGs selected from screened floral development and sex determination-linked genes using the same RNA samples that were used for RNA-Seq from each flower sex type. Briefly, RNA samples were reverse transcribed using iScript cDNA synthesis Kit (Bio-Rad) and used as templates in PCR assays. Two housekeeping genes (actin: CsACT and elongation factor 2: CsEF2) were used as reference genes for normalization. Quantitative PCR (qPCR) was carried out using the StepOne Plus Real-Time PCR system (Applied Bioscience) with a final reaction volume of 10 µl, consisting of 5 µl SYBR premix (Thermo Fisher), 0.6 µM of each primer and 150 ng of cDNA template. The amplification conditions were set withholding stage at 50 °C for 2 min, and 95 °C for 2 min, followed by 50 cycles at 95 °C for 3 s and 60 °C for 30 s, as well as a final melting curve stage at 95 °C for 15 s and 60 °C for 1 min. The primers used in this study are listed in Table S1.

Specific primers were designed with the Primer3 online software (http://bioinfo.ut.ee/primer3-0.4.0/) and ordered online from Eurofins MWG Operon (http://www.operon.com). Primers used in this study are listed in Table 2. Quantitative RT-PCR analysis was carried out by using the SYBR premix (Thermo Scientific Dreieich, Germany) on an iCycler PCR machine (Bio-Rad, Heidelberg, Germany), according to the manufacturer´s instructions. Normalization for cDNA quantity was done using Hprt control primer for each template. The fold change and the normalized values for different mRNAs of Pparγ WT and Pparγ KO were calculated by using the ddCT method. All RT-PCR experiments were performed three times using the total RNA from three distinct isolation experiments. Graphs were made using the GraphPad prism software version 5 and the statistical significance was determined using the unpaired t-test.

Total RNA was extracted from B73 seedling shoots (i.e., aerial parts) using Trizol reagent (Invitrogen). For the qRT-PCR analysis, the extracted total RNA was treated with RQ1 RNase-free DNase (Promega), after which first-strand cDNA was amplified using M-MLV Reverse Transcriptase. The qRT-PCR was completed using the ABI 7500 Real-Time PCR System (Applied Biosystems, USA) and SYBR Premix (Thermo Scientific, USA). A more thorough description of the qRT-PCR procedure is provided in one of our previous publications [11 (link)], and the primers used to amplify the nine genes were designed with the Premier 5 (v5.0) program (see Additional file 1). Two independent experiments were completed, each with three technical replicates. The results of a representative experiment are provided, with data presented as the mean ± SD (n = 3). The extracted total RNA was also used to prepare RNA-seq libraries according to the Illumina Standard mRNA-seq Library Preparation kit (Illumina). The RNA-seq was completed using the Illumina HiSeq 2000 system as previously described [11 (link)]. The RNA-seq experiment (including the library construction) was completed with two biological replicates.

S. aureus ATCC 33591 in MHB was treated with sub-inhibitory concentrations (1/8 MIC, 1/4 MIC, and 1/2 MIC) of TCA for 0.5 h with untreated as a control. Easy-RED BYF RNA was prepared with the Easy-RedByFrNA extraction kit according to the manufacturer’s instructions (iNtRON Biotechnology, Seongnam, Korea). RNA was reverse transcribed into cDNA and was performed by a cDNA synthesis kit (iNtRON Biotechnology) for first-strand cDNA synthesis, in accordance with the manufacturer’s instructions, in order to synthesize the RNA template for qRT-PCR. Primers used are presented in Table 1. A total 20 μL volume was used in the qRT-PCR: 2 μL sample cDNA and 2 μL of primer mix (10 μM), 10 μL of 2 × SYBR premix (Life Technologies, Carlsbad, CA, USA), and 6 μL of nuclease-free water. The PCR was performed by the StepOnePlus real-time PCR system (Applied Biosystems, Courtaboeuf, France).