title: RNA-Seq of Bama Huoma: male flower bud
design: Total RNA for each sample was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. The RNA content and purity of each sample were quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA). RNA integrity was assessed with an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, California, USA) based on an RIN >7.0. DNase I was used to degrade double-stranded and single-stranded DNA contaminants within the RNA samples. The RNA samples were ultimately divided into two groups for library construction and RNA-seq validation. Approximately 5 g of total RNA was subjected to ribosomal RNA depletion according to the manufacturers instructions for the Ribo-Zero rRNA Removal Kit (Illumina, San Diego, USA). After removing ribosomal RNA, the remaining RNA was fragmented into small pieces using divalent cations under high temperature. Then, the cleaved RNA fragments were reverse-transcribed to create cDNA, which was next used to synthesize U-labeled second-stranded DNA with E. coli DNA polymerase I, RNase H and dUTP. An A-base was then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contained a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Single- or dual-index adapters were ligated to the fragments, and size selection was performed with AMPure XP beads. After heat-labile UDG enzyme treatment of the U-labeled second-stranded DNA, the ligated products were amplified by PCR under the following conditions: initial denaturation at 95 C for 3 min; eight cycles of denaturation at 98 C for 15 sec, annealing at 60 C for 15 sec, and extension at 72 C for 30 sec; and a final extension at 72 C for 5 min. The average insert size for the final cDNA library was 300 bp ( 50 bp). Finally, we performed paired-end sequencing on the Illumina NovaSeq 6000 platform