Validating RNA-seq with qRT-PCR Analysis

To validate the results of RNA-seq, quantitative real-time PCR (qRT-PCR) was performed. Nine DEGs (ANR1, ANR2, F3H1, F3H2, DFR1, DFR2, F3′5′H1, ANS1, and ANS2) were selected for qRT-PCR analysis to verify the expression patterns in DCF, LCF, and WCF. The respective qRT-PCR primers are listed in Table S5. Total RNA was extracted from upland cotton fiber using TRIzol Reagent. cDNA synthesis was performed using an RT reagent kit (Tiangen, Beijing, China). qRT-PCR was analyzed in a 20 μL reaction system (including 10 μL SYBR Premix Ex Taq ™ Ⅱ (2×), 2 μL cDNA, and 0.8 μL upstream and downstream primers) and a simple procedure (50 °C for 2 min; 40 cycles at 95 °C for 30 s, 95 °C for 5 s, and 60 °C for 20 s, and a final extension at 72 °C for 10 min). The GhUBQ gene was used as a control, and each sample was repeated 3 times. The relative expression levels were calculated using the 2^−ΔΔCt method [43 (link)].