RNA-Seq of cotton: Seed embryo
A polysaccharide and polyphenol plant total RNA extraction kitwas used to extract RNA (each sample was approximately 100 mg).The extraction process was carried out according to themanufacturers instructions. After RNA extraction, a Nanodropwas used to determine whether the purity (OD260/280),concentration, and nucleic acid absorbance peak of the RNA werenormal. An Agilent 2100 was used to accurately determine theintegrity of the RNA. The indicators included the RIN, 28S/18Sratio, whether the spectral baseline was increased and the presenceof the 5S peak. The concentration of RNA in all the samples was600 ng/ml, and the total amount of RNA was 2 mg. The samplesthat met the criteria were shipped to Beijing Biomarker Biotech Co.,Ltd. (Beijing, China) for sequencing. Oligo (dT) magnetic beadswere used to enrich eukaryotic mRNA through the binding of ATcomplementary pairs to the poly(A) tail of the mRNA. Then,fragmentation buffer was added to fragment the mRNA into shortfragments. The mRNA was used as a template to synthesize firststrand cDNA with random hexamers, followed by the addition ofbuffer, dNTPs, and DNA polymerase I to synthesize second-strandcDNA, which was subsequently synthesized by AMPure. Doublestranded cDNA was purified using XP beads. The purified doublestranded cDNA was end-repaired and A-tailed, and sequencingadapters were ligated. The fragments were subsequently selectedusing AMPure XP beads, after which PCR enrichment wasperformed to obtain the final cDNA library.