Fastking gdna dispelling rt supermix

Liquid samples (1.5 ml) obtained without concentrating the cells were centrifuged (21,000 × g, 10 min at 4°C) to collect cells. Chromosomal DNA was extracted using a DNeasy PowerSoil Pro Kit (QIAGEN, Germany) according to the manufacturer’s instructions. RNA was extracted using an E.Z.N. A Soil RNA Mini Kit (OMEGA, United States). Complementary DNA (cDNA) was synthesized using FastKing gDNA Dispelling RT SuperMix (TianGen Biotech, Beijing, China). The cDNA synthesis reaction was performed with an incubation time of 15 min at 42°C followed by 3 min at 95°C in a DNA Engine Peltier Thermal Cycler (Bio-Rad, CA, United States) (Doğan-Subaşı et al., 2014 (link)).

Intact leaves in the middle of watermelon vines and healthy roots were collected from 10 to 34 days after the pollination stage. The center flesh and seeds were collected and immediately frozen in liquid nitrogen for further use. Three seedlings or fruits were collected for each analysis.
For transcriptome analysis, 12 RNA libraries were built with a TruSeq^TM RNA Sample Prep Kit (Illumina, USA) and sequenced on the Illumina HiSeq 4000 platform using the paired-end 150 bp read mode. Gene expression levels were normalized to transcripts per million reads (TPM) values. The differentially expressed genes (DEGs) (p-adjust < 0.05 and | log2FC | > = 1) between the clnac68 mutant lines and WT lines were selected for further research. The data were analyzed on the free online platform of the Majorbio I-Sanger Cloud Platform (www.i-sanger.com).
For qRT-PCR analysis, cDNA was reverse transcribed with FastKing gDNA Dispelling RT SuperMix (Tiangen Biotech, Beijing, China). qRT-PCR was performed in a LightCycler 480 RT-PCR system (Roche, Switzerland) with specific primers (Supplemental Table S1). The watermelon actin gene Cla97C02G026960 was used as the internal control.

To verify the accuracy and repeatability of the RNA-seq results of DEGs, 10 genes were selected to measure expression levels in different follicles using qRT-PCR. The primers used for amplification of the candidate genes, including anti-Müllerian hormone (AMH), cyclin O (CCNO), cadherin 3 (CDH3), follistatin (FST), and NPC intracellular cholesterol transporter 2 (NPC2), were designed using the Primer-Blast tool in the National Center for Biotechnology Information (NCBI) database and are listed in Supplemental Table S1. The cDNA was synthesized in 2 µg total RNA using a FastKing gDNA Dispelling RT SuperMix (Tiangen, Beijing, China). qRT-PCR was performed on a CFX96TM Real-Time System (Bio-Rad, Hercules, CA, USA) using Real Master Mix SYBR Green I (Tiangen, Beijing, China). The total reaction volume was 20 µL, which comprised 1 µL cDNA, 10 µL 2 × Talent qPCR PreMix, 0.6 µL each of the forward and reverse primers, and 7.8 µL RNase-free water. The thermocycling program was 95 °C for 3 min, followed by 40 cycles of 95 °C for 5 s, 60 °C for 10 s, and 72 °C for 15 s. The $2^{-△△ct}$ method was used to calculate gene expression levels [18 (link)]. Expression levels of coding genes were normalized to those of β-actin.

Total RNA was extracted from 50 mg of rumen epithelial tissue using a FastPure cell/tissue total RNA isolation kit V2 (RC112; Vazyme, Nanjing, China) to detect the expression level of the SCFA transporter genes. The purity and concentration of total RNA were detected using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA). The reaction system for reverse transcription was adding 0.4 μL of RT mix, 1,000 ng of total RNA, and RNase-free ddH₂O to make the volume of 20 μL, using a FastKing gDNA Dispelling RT Super Mix (Tiangen, Beijing, China). The reaction procedure was set as 42°C for 15 min and 95°C for 3 min.
The gDNA were used as the templates for RT-PCR by using a 2×TSINGKE Master qPCR mix (SYBR green I, TSE201; Tsingke, Beijing, China) with an ABI7500 (Thermo Fisher) sequence detector. The reaction system for the PCR was as follows: qPCR mix (10 μL); forward/reverse primer, 0.8 μL (10 μM); 50× ROX reference dye, 0.4 μL; and ddH₂O, up to 20 μL. The PCR procedure followed a previously described protocol (43 (link)). A standard curve method and QuantStudio 7 Flex real-time PCR software (Applied Biosystems, Pleasanton, CA) were used for data analysis according to the 2^−ΔΔCT method (51 (link)). The specific primers for genes SLC16A1, SLC16A3, SLC9A1, SLC9A3, SLC4A2, ATP6V1E1, and ATP1A1 are shown in Table S6 in the supplemental material.

Cells were cultured in six-well plates, total RNA was extracted using the Simply P Total RNA Extraction Kit (BSC52S1; Bioflux, Beijing, China), and cDNA was synthesized using FastKing gDNA Dispelling RT SuperMix (KR118-02; Tiangen Biotech, Beijing, China) [52 (link)]. Real-time PCR was performed using the ABI QuantStudio 5 (Applied Biosystems, Carlsbad, CA, USA) with PowerUp SYBR Green Master Mix (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in a 10 μL reaction system according to the manufacturer’s protocol. The primer sequences of the target genes are indicated in Table 2.