Total rna extraction kit r1200

Total RNA extraction kit (R1200), bought from Solarbio (China), was applied to isolate total RNA from cells. Then, a one-stepSuperRT-PCR mix kit (T2240, Solarbio, China) was applied to conduct the qRT-PCR reaction. Afterward, the signals were examined in a PCR system (EDC-810, Eastwin Life Sciences, Inc.). GAPDH was employed as the normalization control. The relative levels of gene were counted utilizing 2^−ΔΔCT. The primers are displayed in Table 1.

The TM-1 ovules at −3 DPA, −1 DPA, 0 DPA, 1 DPA, 3 DPA and 5 DPA and fibers at 7 DPA, 10 DPA, 15 DPA, 20 DPA and 30 DPA were collected. Three biological repeats were collected in each stage. All samples were immediately frozen in liquid nitrogen and stored at −80 °C. Total RNAs were extracted by the Total RNA Extraction Kit (R1200) (Beijing Solarbio Science & Technology Co. Ltd., Beijing, China) from all the samples for transcriptomic analysis. RNA samples were used as templates for reverse transcription with the PrimeScript RT Reagent kit (Takara, Japan) to obtain cDNA libraries. The cDNA libraries were subjected to 101-cycle paired-end sequencing on an Illumina HiSeq 4000 platform at Berry Genomics (Beijing, China). The data were normalized, and FPKM (Fragments per kilobase of transcript per million) was obtained (Table S1). The GhCOMTs with an FPKM > 1 at least in one stage of the ovule and fiber development were employed for further analysis. The genes were classified into specific and nonspecific expression by the method used in previous research [42 (link)]. The expression heatmaps were visualized by TBtools software [37 (link)].

Plant total RNA was extracted by the Total RNA Extraction Kit (R1200) (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The reverse transcription kit was the PrimeScript™ II 1st Strand cDNA Synthesis Kit (TAKARA, Dalian, China). It was used to reverse the extracted RNA to obtain the first-strand cDNA for transcriptomic and RT qPCR analysis. The expression heatmap was visualized by Tbtools [60 (link)]. The fluorescent quantitative kit was the Taq Pro Universal SYBR qPCR Master Mix (Q712-02) (Vazyme Biotech Co., Ltd, Nanjing China). The data were calculated according to the 2^{−ΔΔ CT} method. The G. hirsutum L. the His3 (GhHis3) gene were used as reference controls [48 (link)]. According to the candidate gene sequence, a relatively specific primer for real-time fluorescence quantitative PCR was designed by Primer3 software, and the amplification product was 150–300 bp. The RTqPCR primers used in this study are shown in Table S23. All primers were synthesized by (Sangon Biotech Co., Ltd, Shanghai, China).