RNA-seq Data Analysis Pipeline

The RNA library was constructed by using the NEBNext ultra directional RNA library prep kit for illumina (New England Biolabs, Ipswich, MA, USA) following the manufacturer's instruction. After the library quality was assessed by Aglient 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA), the next-generation sequencing was performed based on the Illumina HiSeq4000 platform (Illumina Inc., San Diego, CA). Read counts were normalized by TMM (trimmed mean of M values) in the edgeR package [14 (link)]. Then, the read count data were transformed to log2-counts per million (logCPM) for gene expression by the limma-voom package [15 (link)]. The differential gene expression analysis between B-M and A-con cells was carried out by the limma package in R [16 (link), 17 ]. Genes with P < 0.05 and log₂|fold change| ≥ 1 were considered to be significantly different. Cluster analysis for DEGs was performed by gplots in R [18 ].