Novaseq platform hiseq 2500

The total RNA was extracted from the samples using a Plant Total RNA Purification Kit (RC401-01, Vazyme, Nanjing, Jiangsu, China) following the manufacturer’s protocols. To ensure the samples can be used for transcriptome sequencing, the Nanodrop 2000 spectrophotometer (Thermofisher, Waltham, MA, USA), Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA), and Agilent 2100 Bioanalyzer system (Agilent Technologies, Santa Clara, CA, USA) were used to evaluate the purity (OD260/280 ≥ 1.8; OD260/230 ≥ 1.0), concentration (total RNA ≥ 250 ng/µL), and integrity (RIN ≥ 8.0, 28S/18S ≥ 1.5) of RNA samples. DNA library generation and RNA-seq high-throughput sequencing were performed by Majorbio Biotech (Shanghai, China). A total of 18 qualified libraries were sequenced on the Illumina Novaseq platform HiSeq^TM 2500 (Illumina, San Diego, CA, USA), and 150 bp paired-end reads were generated.

The total RNA was extracted from the samples using a Plant Total RNA Purification Kit (TR02–150, GeneMarkbio) following the manufacturer’s instructions. RNA purity was checked using a NanoPhotometer^® spectrophotometer (IMPLEN, Westlake Village, CA, USA). Samples had an RNA Integrity Number (RIN) ≥ 7.0. The quality control was assessed with the Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The mRNA with a polyadenylic acid tail was enriched by connecting oligothymidine magnetic beads, and then the obtained mRNA was randomly interrupted with divalent cations in NEB fragmentation buffer. DNA library generation and RNA-seq high-throughput sequencing were performed by Shenzhen Microeco Biotech (Shenzhen, China). A Qubit2.0 Fluorometer was used for preliminary quantification; the library was diluted to 1.5 ng/uL, and then the Agilent 2100 bioanalyzer was used to detect the insert size of the library. Clustering of the index-coded samples was performed on a cBot Cluster Generation System using a TruSeq PE Cluster Kit v3-cBot-HS according to the manufacturer’s instructions. A total of 8 qualified libraries were sequenced on the Illumina Novaseq platform HiSeqTM 2500 (Illumina, San Diego, CA, USA), and 150 bp paired-end reads were generated.