Transcriptome Analysis of Cotton Diploids

G. arboreum and G. herbaceum were used for the transcriptome sequencing. The RNA-Seq libraries were prepared by a previously described method (Wang et al., 2018 (link)), and a 1% agarose gel was used to check for contamination and degradation of RNA. First, the purity of RNA was analyzed using a Nano Photometer® spectrophotometer (IMPLEN, CA, United States). Next, estimating RNA concentration was performed using a Qubit® RNA Assay Kit and a Qubit® 2.0 Fluorometer (Life Technologies, CA, United States). Finally, RNA integrity was checked using an Agilent Nano 6000 assay kit (Santa Clara, California, United States). Reads counting features (genes, in this case) were performed using HTSeq v0.6.125. Gene lengths and read counts mapped to genes were used to calculate FPKM values (Mortazavi et al., 2008 (link)). The original data was uploaded to NCBI (PRJNA833579).
The differentially expressed genes (DEGs) between diploid and tetraploid were identified with the DESeq R package (Andino et al., 2016 (link)), and Benjamini–Hochberg-adjusted p-values < 0.05 were considered statistically significant (Benjamini and Hochberg, 1995 (link); Anders and Huber, 2010 (link)).