spontaneous nodule

Total RNA from spontaneous nodules, 6 BA induced nodules, and roots was extracted using the RNeasy Plant Mini Kit according to the manufacturer instructions. RNA libraries were sequenced on the DNBSEQ G99 platform as paired-end reads 150 bp in high-output mode. Raw FASTQ files were subjected to quality control using fastp. Clean reads were aligned to the reference genome of M. sativa cv Zhongmu No1 using STAR. Gene expression levels were quantified as raw counts and FPKM fragments per kilobase per million mapped reads using featureCounts. Differentially expressed genes (DEGs) were identified by comparing spontaneous nodules or cytokinin-induced nodules with roots.